U.S. patent application number 11/665903 was filed with the patent office on 2009-07-02 for t-cell stimulatory peptides from the melanoma-associated chondroitin sulfate proteoglycan and their use.
Invention is credited to Gerold Schuler, Erwin Schultz.
Application Number | 20090169573 11/665903 |
Document ID | / |
Family ID | 36072064 |
Filed Date | 2009-07-02 |
United States Patent
Application |
20090169573 |
Kind Code |
A1 |
Schultz; Erwin ; et
al. |
July 2, 2009 |
T-Cell Stimulatory Peptides From The Melanoma-Associated
Chondroitin Sulfate Proteoglycan And Their Use
Abstract
The present invention relates to melanoma-associated chondroitin
sulfate proteoglycan (MCSP) epitopes recognized by T cells,
especially by CD4.sup.+ T lymphocytes (short T-cells) and CD8.sup.+
T cells, on human melanoma cells. In more detail, the present
invention relates to novel T-cell stimulatory tumour antigenic
peptides corresponding to said epitopes (MCSP peptides); to fusion
proteins comprising said MCSP peptides; to the use of said MCSP
peptides, fusion proteins or of the full length MCSP protein itself
or fragments thereof to induce an immune response, especially a
T-cell response; to the use of said MCSP peptides, fusion proteins
or full length MCSP protein itself or fragments thereof to prepare
immune cells, such as mature dendritic cells (DCs) loaded with
anyone of the peptides according to the invention, or
peptide-specific T-cell clones, especially CD4.sup.+ or CD8.sup.+ T
cell clones; to the use of said MCSP peptides, fusion proteins or
MCSP itself or fragments thereof for research and development on/of
a cancer treatment; to the use of said MCSP peptides, fusion
proteins or MCSP itself or fragments thereof for preparing a
medicament for inducing a T cell response in a patient, preferably
for the treatment of cancer, more preferably for the treatment of
melanoma, including cutaneous and ocular melanoma, and other MCSP
expressing tumours such as breast cancer, notably lobular breast
carcinoma, astrocytoma, glioma, glioblastoma, neuroblastoma,
sarcoma and certain types of leukaemia; to the use of said MCSP
peptides, fusion proteins or full length MCSP protein or fragments
thereof for the preparation of a medicament, and a diagnostic agent
for the treatment and prophylaxis as well the diagnosis of an
immune response against tumours; and to the use of said
peptide-specific T-cell clones for diagnosing or treating
cancer.
Inventors: |
Schultz; Erwin; (Amonenburg,
DE) ; Schuler; Gerold; (Spardorf, DE) |
Correspondence
Address: |
Ballard Spahr Andrews & Ingersoll, LLP
SUITE 1000, 999 PEACHTREE STREET
ATLANTA
GA
30309-3915
US
|
Family ID: |
36072064 |
Appl. No.: |
11/665903 |
Filed: |
October 20, 2005 |
PCT Filed: |
October 20, 2005 |
PCT NO: |
PCT/EP05/55430 |
371 Date: |
April 21, 2008 |
Current U.S.
Class: |
424/185.1 ;
424/93.21; 435/29; 435/320.1; 435/325; 435/375; 435/455; 435/6.16;
514/44R; 530/324; 530/325; 530/326; 530/327; 530/328; 530/387.9;
536/23.4; 536/23.5 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 14/4748 20130101 |
Class at
Publication: |
424/185.1 ;
530/328; 530/327; 530/326; 530/325; 530/324; 536/23.5; 536/23.4;
435/320.1; 435/325; 435/375; 435/455; 435/29; 530/387.9; 424/93.21;
514/44; 435/6 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07K 7/00 20060101 C07K007/00; C12N 15/11 20060101
C12N015/11; C12N 15/00 20060101 C12N015/00; C12N 5/06 20060101
C12N005/06; C12N 15/87 20060101 C12N015/87; C12Q 1/02 20060101
C12Q001/02; C07K 16/18 20060101 C07K016/18; A61K 35/12 20060101
A61K035/12; A61K 31/7088 20060101 A61K031/7088; C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 20, 2004 |
EP |
04105199.6 |
Dec 2, 2004 |
EP |
04106250.6 |
Claims
1-23. (canceled)
24. An antigenic T-cell stimulatory peptide (MCSP peptide) which is
derived from the melanoma-associated chondroitin sulfate
proteoglycan (MCSP), has up to 100 amino acid residues, and
comprises at least 8 amino acid residues out of the MCSP segment
represented by amino acid residues 644 to 743 of MCSP of SEQ ID
NO:1, or a functional variant or salt thereof.
25. The MCSP peptide of claim 24, which comprises at least 12 amino
acid residues.
26. The MCSP peptide of claim 24, which is presented by a molecule
selected from the group consisting of HLA-DR, HLA-DQ and
HLA-DP.
27. The MSCP peptide of claim 26, which is presented in full length
by said molecule.
28. The MSCP peptide of claim 26, which is presented in fragmented
form by said molecule.
29. The MCSP peptide of claim 26, which is HLA-DR presented.
30. The MCSP peptide of claim 24, which comprises the amino acid
residues 695 to 705 of SEQ ID NO:1.
31. The MCSP peptide of claim 24, which is selected from the group
consisting of peptides having the following amino acid sequences
(a) LAQGSAMPILPANLSVETNAVGQDVSVLFRVTGALQFGELQK (SEQ ID NO:3); (b)
ETNAVGQDVSVLFRVT (SEQ ID NO:8), (c) VGQDVSVLFRVTGALQ (SEQ ID NO:9),
and fragments of SEQ ID NOs:3, 8 or 9 shortened by up to three
C-terminal and/or N-terminal amino acids.
32. The MCSP peptide of claim 31, which is selected from the group
consisting of SEQ ID NOs:3, 8 and 9 being not shortened on its
termini.
33. A fusion protein comprising as a first domain an MCSP peptide
according to claim 24 (MCSP domain) and at least one second
domain.
34. The fusion protein of claim 33, wherein said at least one
second domain is a protein and/or peptide comprising an endosomal
targeting signal.
35. The fusion protein of claim 33, wherein said at least one
second domain is selected from the group of the human invariable
chain (Ii), a peptide fragment thereof comprising amino acid
residues 1-80, the lysosome-associated membrane protein (LAMP-1)
and DC-LAMP.
36. The fusion protein of claim 33, wherein said MCSP domain and
said at least one second domain are connected directly or through a
linker peptide.
37. A nucleic acid sequence encoding the MCSP peptide of claim
24.
38. A nucleic acid sequence encoding the fusion protein of claim
33.
39. A vector comprising the nucleic acid sequence of claim 37.
40. A vector comprising the nucleic acid sequence of claim 38.
41. A cell transfected or transformed with the vector of claim 39,
and/or comprising a nucleic acid encoding an antigenic T-cell
stimulatory peptide (MCSP peptide) which is derived from the
melanoma-associated chondroitin sulfate proteoglycan (MCSP), has up
to 100 amino acid residues, and comprises at least 8 amino acid
residues out of the MCSP segment represented by amino acid residues
644 to 743 of MCSP of SEQ ID NO:1, or a functional variant or salt
thereof.
42. A cell transfected or transformed with the vector of claim 40,
and/or comprising a nucleic acid encoding a fusion protein
comprising as a first domain an antigenic T-cell stimulatory
peptide (MCSP peptide) which is derived from the
melanoma-associated chondroitin sulfate proteoglycan (MCSP), has up
to 100 amino acid residues, and comprises at least 8 amino acid
residues out of the MCSP segment represented by amino acid residues
644 to 743 of MCSP of SEQ ID NO:1, or a functional variant or salt
thereof (MCSP domain), and at least one second domain.
43. A method to generate stable mature dendritic cells (DCs) loaded
with one or more of the peptides/proteins selected from the group
consisting of (a) one or more of the MCSP peptides and functional
variants of claim 24, (b) one or more fusion proteins comprising as
a first domain an antigenic T-cell stimulatory peptide (MCSP
peptide) which is derived from the melanoma-associated chondroitin
sulfate proteoglycan (MCSP), has up to 100 amino acid residues, and
comprises at least 8 amino acid residues out of the MCSP segment
represented by amino acid residues 644 to 743 of MCSP of SEQ ID
NO:1, or a functional variant or salt thereof (MCSP domain), and at
least one second domain., and (c) the full length MCSP protein of
SEQ ID NO:1 and fragments thereof, which method comprises the
following steps: (i) contacting isolated immature DCs or mature DCs
with one or more of said peptides/proteins (a) to (c) defined
above, to allow for uptake of said peptides/proteins, or contacting
isolated immature DCs or mature DCs with one or more of the nucleic
acid sequence encoding the peptides/proteins (a) to (c) defined
above and/or with one or more vectors comprising nucleic acid
sequences encoding the peptides/proteins (a) to (c) defined above,
to allow for uptake and subsequent expression of the
peptides/proteins in the DC; and (ii) in case of immature DCs,
maturing the DCs obtained in (i) by exposing them to a cytokine
comprising maturation cocktail.
44. A method to generate a T-cell clone specific for one or more of
the peptides/proteins selected from the group consisting of (a) one
or more of the MCSP peptides and functional variants of claim 24,
and (b) the full length MCSP protein of SEQ ID NO:1 and fragments
thereof, which method comprises the following steps: (i) contacting
isolated T-cells with an antigen presenting cell (APC) presenting
anyone of the peptides/proteins (a) to (b) as defined above,
whereby the APC is selected from the group consisting of
B-lymphocytes, macrophages, and DCs; (ii) co-culturing the isolated
T-cells with the APC for at least 30 days, whereby freshly prepared
APCs are added for at least 3 times to the original co-culture, and
the T-cells proliferate; (iii) assessing the ability of the
proliferating T-cells of (ii) to produce cytokines selected from
the group consisting of TNF-.alpha., IFN-.gamma., GM-CSF, IL-2 in
response to the addition of stimulator cells pulsed with anyone of
the peptides/proteins (a) to (b) defined above, whereby the
stimulator cells are selected from the group of autologous or
allogenic immortalized B-cells, DCs, monocytes, and macrophages
pulsed with anyone of the peptides/proteins as defined in (a) to
(b) above; (iv) cloning the TNF-.alpha. and/or IFN-.gamma.
producing T-cells of (iii) by limiting dilution culture in the
presence of autologous or allogenic stimulator cells pulsed with
anyone of the peptides/proteins (a) to (b) as defined above, and
feeder cells, whereby the feeder cells are selected from the group
of allogenic or autologous immortalized B-cells, LG2-EBV and
allogenic or autologous PBMCs; and (v) maintaining the isolated
T-cell clone of step (iv) in the presence of feeder cells in
culture medium comprising a maturation cocktail.
45. The method of claim 44 which is suitable to prepare a CD4.sup.+
or CD8.sup.+ T cell clone.
46. The method of claim 44, wherein the maturation cocktail
comprises interleukin-2 (IL-2), interleukin-7 (IL-7) and
phytohemagglutinin (PHA) 13.
47. A mature DC loaded with one or more of the peptides/proteins
selected from the group consisting of (a) one or more of the MCSP
peptides and functional variants of claim 24, (b) one or more
fusion proteins comprising as a first domain an antigenic T-cell
stimulatory peptide (MCSP peptide) which is derived from the
melanoma-associated chondroitin sulfate proteoglycan (MCSP), has up
to 100 amino acid residues, and comprises at least 8 amino acid
residues out of the MCSP segment represented by amino acid residues
644 to 743 of MCSP of SEQ ID NO:1, or a functional variant or salt
thereof (MCSP domain), and at least one second domain, and (c) the
full length MCSP protein of SEQ ID NO:1 and fragments thereof.
48. A T-cell clone specific for one or more of the
peptides/proteins selected from (a) one or more of the MCSP
peptides and functional variants of claim 24, (b) one or more
fusion proteins comprising as a first domain an antigenic T-cell
stimulatory peptide (MCSP peptide) which is derived from the
melanoma-associated chondroitin sulfate proteoglycan (MCSP), has up
to 100 amino acid residues, and comprises at least 8 amino acid
residues out of the MCSP segment represented by amino acid residues
644 to 743 of MCSP of SEQ ID NO:1, or a functional variant or salt
thereof (MCSP domain), and at least one second domain, and (c) the
full length MCSP protein of SEQ ID NO:1 and fragments thereof.
49. The T-cell clone of claim 48 which is a CD4.sup.+ or CD8.sup.+
T cell clone.
50. An antibody specific for the MCSP peptide of claim 24.
51. An antibody specific for the fusion protein of claim 33.
52. A pharmaceutical or diagnostic composition comprising one or
more of the functional components selected from the group
consisting of: the MCSP peptides and functional variants of claim
24; a fusion protein comprising as a first domain an MCSP peptide
according to claim 24 (MCSP domain) and at least one second domain;
a nucleic acid sequence encoding the MCSP peptide of claim 24; a
nucleic acid sequence encoding a fusion protein comprising as a
first domain an MCSP peptide according to claim 24 (MCSP domain)
and at least one second domain; a vector comprising a nucleic acid
sequence encoding the MCSP peptide of claim 24; a vector comprising
a nucleic acid sequence encoding a fusion protein comprising as a
first domain an MCSP peptide according to claim 24 (MCSP domain)
and at least one second domain; a cell transfected or transformed
with a vector comprising a nucleic acid sequence encoding the MCSP
peptide of claim 24; a cell transfected or transformed with a
vector comprising a nucleic acid sequence encoding a fusion protein
comprising as a first domain an MCSP peptide according to claim 24
(MCSP domain) and at least one second domain; a mature DC loaded
with one or more of the peptides/proteins selected from the group
consisting of (a) one or more of the MCSP peptides and functional
variants of claim 24, (b) one or more fusion proteins comprising as
a first domain an antigenic T-cell stimulatory peptide (MCSP
peptide) which is derived from the melanoma-associated chondroitin
sulfate proteoglycan (MCSP), has up to 100 amino acid residues, and
comprises at least 8 amino acid residues out of the MCSP segment
represented by amino acid residues 644 to 743 of MCSP of SEQ ID
NO:1, or a functional variant or salt thereof (MCSP domain), and at
least one second domain, and (c) the full length MCSP protein of
SEQ ID NO:1 and fragments thereof; a T-cell clone specific for one
or more of the peptides/proteins selected from (a) one or more of
the MCSP peptides and functional variants of claim 24, (b) one or
more fusion proteins comprising as a first domain an antigenic
T-cell stimulatory peptide (MCSP peptide) which is derived from the
melanoma-associated chondroitin sulfate proteoglycan (MCSP), has up
to 100 amino acid residues, and comprises at least 8 amino acid
residues out of the MCSP segment represented by amino acid residues
644 to 743 of MCSP of SEQ ID NO:1, or a functional variant or salt
thereof (MCSP domain), and at least one second domain, and (c) the
full length MCSP protein of SEQ ID NO:1 and fragments thereof; an
antibody specific for the MCSP peptide of claim 24; and an antibody
specific for a fusion protein comprising as a first domain an MCSP
peptide according to claim 24 (MCSP domain) and at least one second
domain; and a pharmaceutically or diagnostically acceptable
carrier.
53. The composition of claim 52, which is a vaccine further
comprising an adjuvant.
54. A method for diagnosing and/or monitoring a disorder
characterized by the expression of one or more of the MCSP peptides
of claim 24, the full length MCSP protein of SEQ ID NO:1 and
fragments thereof, which method comprises the following steps:
contacting a biological sample isolated from a subject having or
suspected to have said disorder, with an agent that is specific for
anyone of the MCSP peptides of claim 24, the full length MCSP
protein of SEQ ID NO:1 and fragments thereof; and determining the
interaction between the agent and the peptide.
55. A method for preventing or treating cancer in a patient, which
method comprises administering to the patient an effective amount
of one or more of the agents selected from the group consisting of:
the MCSP peptides and/or the functional variants of claim 24; a
fusion protein comprising as a first domain an MCSP peptide
according to claim 24 (MCSP domain) and at least one second domain;
a nucleic acid sequence encoding the MCSP peptide of claim 24; a
nucleic acid sequence encoding a fusion protein comprising as a
first domain an MCSP peptide according to claim 24 (MCSP domain)
and at least one second domain; a vector comprising a nucleic acid
sequence encoding the MCSP peptide of claim 24; a vector comprising
a nucleic acid sequence encoding a fusion protein comprising as a
first domain an MCSP peptide according to claim 24 (MCSP domain)
and at least one second domain, the full length MCSP protein of SEQ
ID NO:1 and fragments thereof, nucleic acid sequences encoding the
full length MCSP protein of SEQ ID NO:1 or fragments thereof, and
vectors comprising a nucleic acid sequence encoding the full length
MCSP protein of SEQ ID NO:1 or fragments thereof; a cell
transfected or transformed with a vector comprising a nucleic acid
sequence encoding the MCSP peptide of claim 24; a cell transfected
or transformed with a vector comprising a nucleic acid sequence
encoding a fusion protein comprising as a first domain an MCSP
peptide according to claim 24 (MCSP domain) and at least one second
domain; a mature DC loaded with one or more of the
peptides/proteins selected from the group consisting of (a) one or
more of the MCSP peptides and functional variants of claim 24, (b)
one or more fusion proteins comprising as a first domain an
antigenic T-cell stimulatory peptide (MCSP peptide) which is
derived from the melanoma-associated chondroitin sulfate
proteoglycan (MCSP), has up to 100 amino acid residues, and
comprises at least 8 amino acid residues out of the MCSP segment
represented by amino acid residues 644 to 743 of MCSP of SEQ ID
NO:1, or a functional variant or salt thereof (MCSP domain), and at
least one second domain, and (c) the full length MCSP protein of
SEQ ID NO:1 and fragments thereof; a T-cell clone specific for one
or more of the peptides/proteins selected from (a) one or more of
the MCSP peptides and functional variants of claim 24, (b) one or
more fusion proteins comprising as a first domain an antigenic
T-cell stimulatory peptide (MCSP peptide) which is derived from the
melanoma-associated chondroitin sulfate proteoglycan (MCSP), has up
to 100 amino acid residues, and comprises at least 8 amino acid
residues out of the MCSP segment represented by amino acid residues
644 to 743 of MCSP of SEQ ID NO:1, or a functional variant or salt
thereof (MCSP domain), and at least one second domain, and (c) the
full length MCSP protein of SEQ ID NO:1 and fragments thereof; an
antibody specific for the MCSP peptide of claim 24; and an antibody
specific for a fusion protein comprising as a first domain an MCSP
peptide according to claim 24 (MCSP domain) and at least one second
domain; a pharmaceutical composition comprising an of the above and
a pharmaceutical composition comprising any of the above and
further comprising an adjuvant.
56. The method of claim 55, wherein the cancer is selected from the
group consisting of melanoma, including cutaneous and ocular
melanoma, and MCSP expressing tumours, including breast cancer,
lobular breast carcinoma, astrocytoma, glioma, glioblastoma,
neuroblastoma, sarcoma and certain types of leukaemia.
57. A method for diagnosing cancer which comprises utilizing a
diagnostic marker selected from the group consisting of the MCSP
peptides or variants of claim 24, a fusion protein comprising as a
first domain an MCSP peptide according to claim 24 (MCSP domain)
and at least one second domain, a nucleic acid sequence encoding
the MCSP peptide of claim 2, a nucleic acid sequence encoding a
fusion protein comprising as a first domain an MCSP peptide
according to claim 24 (MCSP domain) and at least one second domain,
a vector comprising a nucleic acid sequence encoding the MCSP
peptide of claim 24, a vector comprising a nucleic acid sequence
encoding a fusion protein comprising as a first domain an MCSP
peptide according to claim 24 (MCSP domain) and at least one second
domain; the full length MCSP protein of SEQ ID NO:1 and fragments
thereof, a nucleic acid sequence encoding the full length MCSP
protein of SEQ ID NO:1 or fragments thereof, and vectors comprising
nucleic acid sequences encoding the full length MCSP protein of SEQ
ID NO:1 or fragments thereof.
58. The method of claim 57, wherein the cancer is selected from the
group consisting of melanoma, including cutaneous and ocular
melanoma, and MCSP expressing tumours including breast cancer,
lobular breast carcinoma, astrocytoma, glioma, glioblastoma,
neuroblastoma, sarcoma and certain types of leukaemia.
59. A method for inducing a T cell response in a patient, which
method comprises administering to the patient an effective amount
of one or more of the agents selected from the group consisting of:
the MCSP peptides and/or the functional variants of claim 24, a
fusion protein comprising as a first domain an MCSP peptide
according to claim 24 (MCSP domain) and at least one second domain,
a nucleic acid sequence encoding the MCSP peptide of claim 24, a
nucleic acid sequence encoding a fusion protein comprising as a
first domain an MCSP peptide according to claim 24 (MCSP domain)
and at least one second domain; a vector comprising a nucleic acid
sequence encoding the MCSP peptide of claim 24, a vector comprising
a nucleic acid sequence encoding a fusion protein comprising as a
first domain an MCSP peptide according to claim 24 (MCSP domain)
and at least one second domain, the full length MCSP protein of SEQ
ID NO:1 and fragments thereof, nucleic acid sequences encoding the
full length MCSP protein of SEQ ID NO:1 or fragments thereof, and
vectors comprising a nucleic acid sequence encoding the full length
MCSP protein of SEQ ID NO:1 or fragments thereof.
Description
[0001] The present invention relates to melanoma-associated
chondroitin sulfate proteoglycan (MCSP) epitopes recognized by T
cells, especially by CD4.sup.+ T lymphocytes (short T-cells) and
CD8.sup.+ T cells, on human melanoma cells. In more detail, the
present invention relates to novel T-cell stimulatory tumour
antigenic peptides corresponding to said epitopes (MCSP peptides);
to fusion proteins comprising said MCSP peptides; to the use of
said MCSP peptides, fusion proteins or of the full length MCSP
protein itself or fragments thereof to induce an immune response,
especially a T-cell response; to the use of said MCSP peptides,
fusion proteins or full length MCSP protein itself or fragments
thereof to prepare immune cells, such as mature dendritic cells
(DCs) loaded with anyone of the peptides according to the
invention, or peptide-specific T-cell clones, especially CD4.sup.+
or CD8.sup.+ T cell clones; to the use of said MCSP peptides,
fusion proteins or MCSP itself or fragments thereof for research
and development on/of a cancer treatment; to the use of said MCSP
peptides, fusion proteins or MCSP itself or fragments thereof for
preparing a medicament for inducing a T cell response in a patient,
preferably for the treatment of cancer, more preferably for the
treatment of melanoma, including cutaneous and ocular melanoma, and
other MCSP expressing tumours such as breast cancer, notably
lobular breast carcinoma, astrocytoma, glioma, glioblastoma,
neuroblastoma, sarcoma and certain types of leukaemia; to the use
of said MCSP peptides, fusion proteins or full length MCSP protein
or fragments thereof for the preparation of a medicament, and a
diagnostic agent for the treatment and prophylaxis as well the
diagnosis of an immune response against tumours; and to the use of
said peptide-specific T-cell clones for diagnosing or treating
cancer.
BACKGROUND
[0002] During the last two decades there has been considerable
interest in the biology and pathophysiology of human malignant
melanoma, in particular because of the poor prognosis and
increasing incidence of this disease. The fatal nature of
metastasizing human cutaneous melanoma which is attributable to
poor response to conventional radiation and chemotherapy, has
prompted a growing interest in alternative approaches to that
disease. One approach was to look for the expression of tumour
associated proteins which can be used as targets for immunotherapy.
These proteins which are either newly expressed, mutated or
overexpressed in tumours, can be utilized for therapy purposes or
as diagnostic markers. The human melanoma-associated chondroitin
sulfate proteoglycan (MCSP) is a tumour-associated protein. It is
uniformly expressed on >90% of human malignant melanoma tissues
and cultured cells, but shows only a limited expression pattern in
normal tissue (Bumol & Reisfeld, PNAS 79, 1245-1249 (1982);
Bumol et al., J. Biol. Chem. 267, 12733-12741 (1984); Bumol et al.,
Adv. Exp. Med. Biol. 172, 455-470 (1984); Harper et al., J. Biol.
Chem. 261, 3600-3606 (1986)). The core protein consists of 2322
amino acids (SEQ ID NO:1), encompassing a large extracellular
domain, a hydrophobic transmembrane region, and a relatively short
cytoplasmic tail (Pluschke et al., PNAS 93, 9710-9715 (1996) and WO
97/13855). MCSP exists in cells as a unique
glycoprotein-proteoglycan complex, with a 250 kDa core glycoprotein
to which, via serine residues, the larger than 450 kDa proteoglycan
component is attached. Multiple Northern blots with an
MCSP-specific probe revealed a strong hybridization signal only
with melanoma cells and not with other human cancer cells or a
variety of human fetal and adult tissues (Pluschke et al., PNAS 93,
9710-9715 (1996)).
[0003] MCSP is a cell-surface antigen that has been implicated in
the growth and invasion of melanoma tumours. It was shown that
stimulated MCSP recruits an adaptor protein important in tumour
cell motility and invasion and participates directly in the signal
transduction process crucial for the adhesion and extravasation of
tumour cells, and therefore MCSP appears to be relevant for
melanoma invasion and metastasis (Iida et al., J. Biol. Chem. 276,
18786-18794 (2001); Burg et al., J. Cell. Physiol. 177, 299-312
(1998); Eisenmann et al., Nat. Cell Biol. 1, 507-513 (1999)).
Monoclonal antibodies against MCSP have been available for a long
time. They were obtained by immunizing mice with a plasma
membrane-enriched fraction from human malignant melanoma cells and
subsequent generation of hybridomas (Harper et al., Hybridoma 1,
423-432 (1984); Harper et al., J. Immunol. 132, 2096-2104 (1984)).
More recently two antimelanoma MCSP-specific immunoconjugates
containing a human single-chain Fv (scFv) targeting domain
conjugated to the Fc effector domain of human IgG1 were
synthesized. The Fc effector domain of the immunoconjugates binds
natural killer (NK) cells and also the C1q protein that initiates
the complement cascade, both triggering a powerful cytolytic
response against the targeted tumour cells, resulting in the lysis
of the melanoma cells (Wang et al., PNAS 96, 1627-1632 (1999)). It
is noteworthy that the scFv targeting domains originally were
isolated as melanoma-specific clones from a scFv fusion-phage
library, derived from the antibody repertoire of a vaccinated
melanoma patient (Abdel-Wahab et al., Cancer 80, 401-412 (1997)).
However, the promising in vitro results did not hold up in vivo,
and the antibody or antibody fragment based approaches towards
melanoma have so far not lead to convincing results in the
treatment of melanoma (Oldham et al., J. Clin. Oncol. 11:1235-1244
(1984); Abrams et al., in "Monoclonal Antibodies and Cancer
Therapy" p. 233 (1985), Reisfeld and Sell eds., New York).
[0004] Furthermore, WO 97/13855 utilizes MCSP as an antigen for an
active specific immune response for generating a humoral, i.e.
antibody response. However, up to now a T-cell inducing property
has not been reported for MCSP.
[0005] T-cell based approaches towards the treatment of melanoma
may hold more promise. A T-cell response requires that T-cells
recognize and interact with complexes of cell surface molecules,
referred to as human leukocyte antigen (HLA), or major
histocompatibility complexes (MHCs), and peptides which are bound
to said surface molecules. The peptides are derived from larger
protein molecules which are processed by the cells which also
present the HLA/MHC molecule. The HLA-associated peptides are
short, encompassing 9-25 amino acids (Kropshofer & Vogt,
Immunol. Today 18:77-82 (1997); Male et al., Advanced Immunology,
chapters 6-10, J.P. Lipincott Company (1987)). They are
indispensable for mounting an adaptive immune response as they
activate the T-cells. The lack of T-cell recognition of peptides
derived from tumour-specific antigens contributes to immune evasion
and progressive growth of tumours (Boon et al., Ann. Rev. Immunol.
12, 337-265 (1994)).
[0006] With regard to their function, two classes of HLA-peptide
complexes can be distinguished (Germain, Ann. N.Y. Acad. Sci. 754,
114-25 (1995)). HLA class I-peptide complexes can be expressed by
almost all nucleated cells in order to attract
CD8.sup.+-cytotoxic/cytolytic T cells (CTLs) which lyse cells that
present the appropriate antigen, such as tumour cells or virus
infected cells and thus play an essential role in eradicating said
cells. HLA class II-peptide complexes are constitutively expressed
only on so-called antigen presenting cells (APCs), such as B
lymphocytes (short B-cells), macrophages or DCs. Of particular
importance are DCs, which have the capacity to prime CD4.sup.+-T
helper cells (Banchereau & Steinmann, Nature 392, 245-254
(1998)). CD4.sup.+-T helper cells secrete cytokines to stimulate
macrophages and antigen producing B-cells, which present the
appropriate antigen by HLA class II molecules on their surface.
Moreover, DC can be licensed to activate optimally cytotoxic
CD8.sup.+-T cells. This is accomplished through prior interaction
of their HLA class II peptide complexes with CD4.sup.+-T helper
cells (Ridge et al., Nature 393, 474-478 (1998)). CD4.sup.+ T cell
help is therefore crucial for the induction and maintenance of
strong CTL responses. Moreover, CD4.sup.+ T cells produce huge
amounts of IF-.gamma. which has been shown to inhibit angiogenesis
by tumours. In addition, cytokines secreted by CD4.sup.+ T cells
can recruit other immune cells with antitumour activity such as
macrophages and neutrophils to the tumour site. Finally, CD4.sup.+
T cells can directly recognize and lyse HLA class II expressing
tumour cells such as melanoma cells.
[0007] The apparent pivotal role of DCs in initiating immune
responses has stimulated attempts to exploit DCs as vaccines, in
particularly against cancer (Dallal & Lotze, Curr. Opin.
Immunol. 12, 583-588 (2000); Orsini et al., Br. J. Haematol. 125,
720-728 (2004); Akiyama et al., Anticancer Res. 24, 571-577
(2004)). A key advance was the invention of techniques for
differentiation of DCs in vitro from different sources including
peripheral blood, e.g. adherent monocytes, or bone marrow-derived
CD34.sup.+ stem cell precursors (EP 633929; EP 914415; EP 922758;
EP 1412483; EP 1311658). DC differentiated and activated in vitro
can be used for vaccination of cancer patients after co-culture
with tumour cell-derived antigens or by employing analogous
techniques. Pilot DC vaccination studies have successfully induced
specific anticancer responses (Timmermann & Levy, Ann. Rev.
Med. 50, 507-529 (1999); Yu et al., Cancer Res., 64, 4973-4979
(2004); Barbuto et al., Cancer Immunol. Immunother., Epub ahead of
print, (Jun. 4, 2004); Avigan et al., Clin. Cancer Res. 10,
4699-4708 (2004); Banchereau et al., Cancer Res. 61, 6451-6458
(2001); Chang et al., Clin. Cancer Res. 8, 1021-1032 (2002); Fong
et al., J. Immunol. 166, 4254-4259 (2001); Iwashita et al., Cancer
Immunol. Immunother. 52, 155-161, Epub Feb. 6, 2003 (2003); Marten
et al., Cancer Immunol. Immunother. 51, 637-644, Epub Oct. 3, 2002
(2002); Nestle et al., Nature Medicine 7, 761-765 (2001); Nestle et
al., Nature Med. 4, 328-332 (1998)).
[0008] Vaccines based on the identification of tumour antigens
include DCs primed with naked DNA, recombinant adeno- or vaccinia
viruses, natural or recombinant proteins purified from the
respective tumour cells or synthetic analogues of tumour peptides.
The advantage of pulsing/loading DCs with antigenic tumour peptides
rather than with genetic or protein precursors is that peptides can
directly be loaded onto HLA molecules of DCs without further
processing.
[0009] During the past decade, numerous peptides derived from
tumour specific proteins and restricted by HLA class I molecules
have been identified. In several clinical pilot vaccination
studies, DCs from melanoma patients were pulsed with cocktails of
melanoma peptides which, as yet, were exclusively HLA class I
restricted (Nestle et al., Nature Med. 4, 328-332 (1998); Thurner,
et al., J. Exp. Med. 190, 1669-1678 (1999)). However, there is
increasing evidence that the efficacy and longevity of CTL
responses against tumours can be increased by the recruitment of
HLA class II-restricted T-helper cells.
[0010] Knowledge of HLA class II-restricted cancer antigens
recognized by CD4.sup.+ T helper cells lags behind the
identification of class I-restricted antigens (Wang, Trends in
Immunol. 22, 269-276 (2001)). One reason is that transfection of
cDNA libraries from tumour cells into target cells followed by
usage of anti-tumour T-cells to identify the appropriate
transfectants and antigenic epitopes--a method successfully
employed with HLA class I molecules--is not effective because the
encoded proteins do not travel to the HLA class II pathway in
APCs.
[0011] An example for HLA class II presented human
tumour-associated peptides are those derived from melanoma antigen
(MAGE)-encoding genes, such as the MAGE-A3 peptides (WO 00/20581
and U.S. Pat. No. 6,716,809) presented by HLA-DR molecules or
HLA-DP4. These peptides have already been used in clinical studies
demonstrating that even in advanced melanoma patients strong
CD4.sup.+ T-cell responses can be induced against melanoma cells.
However, only 69% of melanomas express MAGE-A3 (Gaugler et al., J.
Exp. Med. 179, 921-930 (1994)). It is also uncertain which role the
MAGE-protein family, in particular MAGE-A3 plays during tumour
development. An ideal target antigen (i.e. tumour associated
protein) is widely expressed in melanomas (preferably in more than
90%), and its role during tumour development is defined: it plays a
functional role in tumour development and formation of metastasis.
The use of such target antigen reduces the risk of antigen loss
considerably. MAGE-A3 is currently the best tumour antigen found in
terms of clinical studies, i.e. it counts as gold standard for the
development of improved tumour antigens. An ideal candidate for the
development of melanoma-specific therapies however, would have a
higher expression rate than MAGE-A3, i.e. it should be at least
expressed in 90% of the tumours, in particular melanomas.
Furthermore, an ideal antigen would be functionally important for
the tumour cells as it has been shown for MCSP but not for
MAGE-A3.
[0012] More recently HLA/MHC class II presented antigenic peptides
derived from the translation factor eIF-4A, the
IFN-.gamma.-inducible protein p78, the cytoskeletal protein
vimentin and the iron-binding surface protein melanotransferrin
have been described (WO 2004/031230). By contacting DCs in vitro
with necrotic melanoma cells under conditions stimulating antigen
uptake, the DCs were antigen-loaded, and from the mature DCs the
antigen-loaded MHC class II molecules were purified in order to
isolate and identify the associated antigenic peptide. The peptides
identified are also all found in other tissues than malignant
growth. Vimentin, for example, a so-called intermediate filament
protein, is most widely distributed, being present in the cytoplasm
of many animal/human cells of mesodermal origin, including
fibroblasts, endothelial cells, and white blood cells. In addition,
many cells express it transiently during development. The
translation factor eIF-4A is part of the translation machinery
found in every single cell of an organism. The IFN-gamma-inducible
protein p78, also called MxB, is produced by healthy cells in
response to the virally induced presence of interferon.
Melanotransferrin was one of the first surface marker proteins
associated with human melanoma (Hellstrom et al., Int. J. Cancer
31, 553-555 (1983)). However it is also expressed in non-malignant
cells, as an analysis of 50 tissues showed, and in particular it
was found in the salivary glands, endothelial cells in the liver
and brain and the sweat gland ducts (Richardson, Eur. J. Biochem.
267, 1290-1298 (2000)).
SUMMARY OF THE INVENTION
[0013] In view of the above, it is desirable to obtain T-cell
stimulatory, HLA class II-presented peptides from a protein which
is strongly expressed in melanoma cells. Surprisingly it was found,
that such peptides can be isolated from a specific amino acid
region from MCSP, which is expressed in more than 90% of all
melanomas. Since MCSP functions in adhesion, invasion and
metastasis of melanoma are known, it represents an ideal candidate
for the development of immunological anti-tumour/anti-melanoma
therapies. The present invention provides peptides suitable for
various aspects of cancer immunotherapy, including new vaccines and
immunodiagnostic agents.
[0014] Furthermore, the present invention provides the use of MCSP,
or of fragments, derivatives or variants thereof, as T-cell
inducing agent. This agent can be used in cancer immunotherapy,
especially as anti-cancer vaccine.
[0015] In more detail, the present invention provides:
(1) An antigenic T-cell stimulatory peptide (hereinafter "MCSP
peptide") which is derived from the melanoma-associated chondroitin
sulfate proteoglycan (MCSP), has up to 100 amino acid residues, and
comprises at least 8 amino acid residues out of the MCSP segment
represented by amino acid residues 644 to 743 or 1270 to 1300 of
MCSP of SEQ ID NO:1, or a functional variant or salt thereof; (2) a
fusion protein comprising as a first domain an MCSP peptide as
defined in (1) above (MCSP domain) and at least one second domain;
(3) a nucleic acid sequence encoding anyone of the MCSP peptides
and fusion proteins as defined in (1) and (2) above; (4) a vector
comprising the nucleic acid sequence as defined in (3) above; (5)
an isolated cell transfected or transformed with the vector as
defined in (4) above and/or comprising the nucleic acid as defined
in (3) above, whereby the cell is preferably selected from the
group consisting of mammalian cells, preferably human or murine
cells, more preferably primary cells such as melanoma cells,
T-cells, antigen presenting cells including DCs, macrophages and
B-cells, microorganism cells such as fungal cells, yeast cells and
bacterial cells etc., insect cells and plant cells; (6) use of
anyone of the peptides and fusion proteins as defined in (1) and
(2) above for research on and development of medicaments for cancer
treatment, preferably for a treatment of melanoma and other MCSP
expressing tumours such as breast cancer, notably lobular breast
carcinoma, astrocytoma, glioma, glioblastoma, neuroblastoma,
sarcoma and certain types of leukaemia; (7) a method to generate
stable mature dendritic cells (DCs) loaded with one or more of the
peptides/proteins selected from (a) one or more of the MCSP
peptides and functional variants as defined in (1) above, (b) one
or more of the fusion proteins as defined in (2) above, and (c) the
full length MCSP protein of SEQ ID NO:1 and fragments thereof,
which method comprises the following steps: [0016] (i) contacting
isolated immature DCs or mature DCs with one or more of said
peptides/proteins (a) to (c) defined above, to allow for uptake of
said peptides/proteins, or [0017] contacting isolated immature DCs
or mature DCs with one or more of the nucleic acid sequence
encoding the peptides/proteins (a) to (c) defined above and/or with
one or more vectors comprising nucleic acid sequences encoding the
peptides/proteins (a) to (c) defined above, to allow for uptake and
subsequent expression of the peptides/proteins in the DC; and
[0018] (ii) in case of immature DCs, maturing the DCs obtained in
(i) by exposing them to a cytokine comprising maturation cocktail;
(8) a method to generate a T-cell clone, preferably a CD4.sup.+ or
CD8.sup.+ T cell clone, specific for one or more of the
peptides/proteins selected from (a) one or more of the MCSP
peptides and functional variants as defined in (1) above, (b) one
or more of the fusion proteins as defined in (2) above, and (c) the
full length MCSP protein of SEQ ID NO:1 and fragments thereof,
which method comprises the following steps: [0019] (i) contacting
isolated T-cells, preferably isolated CD4.sup.+ or CD8.sup.+ T
cells, with an antigen presenting cell (APC) presenting anyone of
the peptides/proteins (a) to (c) as defined above, whereby the APC
is selected from the group of B-lymphocytes, macrophages, and/or
DCs, preferentially is a DC generated by the method of (7) above;
[0020] (ii) co-culturing the isolated T-cells with the APC for at
least 30 days, whereby freshly prepared APCs are added for at least
3 times to the original co-culture, and the T-cells proliferate;
[0021] (iii) assessing the ability of the proliferating T-cells of
(ii) to produce cytokines selected from the group of TNF-.alpha.,
IFN-.gamma., GM-CSF, IL-2 in response to the addition of stimulator
cells pulsed with anyone of the peptides/proteins (a) to (c)
defined above, whereby the stimulator cells are selected from the
group of autologous or allogenic immortalized B-cells, DCs,
monocytes, and macrophages pulsed with anyone of the
peptides/proteins as defined in (a) to (c) above; [0022] (iv)
cloning the TNF-.alpha. and/or IFN-.gamma. producing T-cells of
(iii) by limiting dilution culture in the presence of autologous or
allogenic stimulator cells pulsed with anyone of the
peptides/proteins (a) to (c) as defined above, and feeder cells,
whereby the feeder cells are selected from the group of allogenic
or autologous immortalized B-cells, LG2-EBV and allogenic or
autologous PBMCS; and [0023] (v) maintaining the isolated T-cell
clone of step (iv) in the presence of feeder cells in culture
medium comprising a maturation cocktail, preferably comprising
interleukin-2 (IL-2), interleukin-7 (IL-7) and phytohemagglutinin
(PHA); (9) a mature DC loaded with one or more of the
peptides/proteins selected from (a) one or more of the MCSP
peptides and functional variants as defined in (1) above, (b) one
or more of the fusion proteins as defined in (2) above, and (c) the
full length MCSP protein of SEQ ID NO:1 and fragments thereof,
preferably the mature loaded DC is a DC obtainable by the method as
defined in (7) above; (10) a T-cell clone, preferably a CD4.sup.+
or CD8.sup.+ T cell clone, specific for one or more of the
peptides/proteins selected from (a) one or more of the MCSP
peptides and functional variants as defined in (1) above, (b) one
or more of the fusion proteins as defined in (2) above, and (c) the
full length MCSP protein of SEQ ID NO:1 and fragments thereof,
preferably said T-cell clone is a T cell clone obtainable by the
method as defined in (8) above; (11) an antibody specific for
anyone of the MCSP peptides, functional variants and fusion
proteins as defined in (1) and (2) above; (12) a pharmaceutical or
diagnostic composition comprising one or more of the MCSP peptides
and fusion proteins as defined in (1) and (2) above, the nucleic
acid sequences as defined in (3) above, the vectors as defined in
(4) above, the transfected and/or transformed cells as defined in
(5) above, the loaded mature DCs as defined in (9) above, the
T-cell clones as defined in (10) above and/or the antibodies as
defined in (11) above, and a pharmaceutically or diagnostically
acceptable carrier; (13) use of one or more of the MCSP peptides
and fusion proteins as defined in (1) and (2) above, the nucleic
acid sequences as defined in (3) above, the vectors as defined in
(4) above, the full length MCSP protein of SEQ ID NO:1 and
fragments thereof, nucleic acids encoding the full length MCSP
protein of SEQ ID NO:1 or fragments thereof, and vectors comprising
nucleic acid sequences encoding the full length MCSP protein of SEQ
ID NO:1 or fragments thereof [0024] (i) for the preparation of
immune cells, preferably for the preparation of artificial APCs,
mature loaded DCs, T-cell clones, B-cells secreting the antibodies
as defined in (11) above and/or hybridomas secreting said
antibodies; and/or [0025] (ii) as a diagnostic marker for cancer,
preferably as a diagnostic marker for melanoma, including cutaneous
and ocular melanoma, and other MCSP expressing tumours such as
breast cancer, notably lobular breast carcinoma, astrocytoma,
glioma, glioblastoma, neuroblastoma, sarcoma and certain types of
leukaemia; and/or [0026] (iii) for the preparation of a medicament
for preventing, treating and/or diagnosing cancer, preferably for
preventing, treating and/or diagnosing melanoma, including
cutaneous and ocular melanoma, and other MCSP expressing tumours
such as breast cancer, notably lobular breast carcinoma,
astrocytoma, glioma, glioblastoma, neuroblastoma, sarcoma and
certain types of leukaemia; [0027] (iv) for the manufacturing of a
medicament inducing a T cell response in a patient; (14) use of the
transfected and/or transformed cells as defined in (5) above, the
loaded mature DCs as defined in (9) above, the T-cell clones as
defined in (10) above and/or the antibodies as defined in (11)
above for the preparation of a medicament for preventing, treating
and/or diagnosing cancer, preferably for preventing, treating
and/or diagnosing melanoma, including cutaneous and ocular
melanoma, and other MCSP expressing tumours such as breast cancer,
notably lobular breast carcinoma, astrocytoma, glioma,
glioblastoma, neuroblastoma, sarcoma and certain types of
leukaemia; (15) use of one or more of the MCSP peptides and fusion
proteins as defined in (1) and (2) above, the nucleic acid
sequences as defined in (3) above, the vectors as defined in (4)
above, for the manufacturing of a medicament stimulating the
production of protective antibodies and/or immune cells; (16) a
method for diagnosing and/or monitoring a disorder characterized by
the expression of one or more of the MCSP peptides and fusion
proteins as defined in (1) and (2) above, the full length MCSP
protein of SEQ ID NO:1 and fragments thereof, comprising the
following steps: (i) contacting a biological sample isolated from a
subject having or suspected to have said disorder, with an agent
that is specific for anyone of the MCSP peptides and fusion
proteins as defined in (1) and (2) above, the full length MCSP
protein of SEQ ID NO:1 and fragments thereof, preferably the agent
being a T-cell clone as defined in (10) above or an antibody as
defined in (11) above, and (ii) determining the interaction between
the agent and the peptide; (17) a method for preventing or treating
cancer, in particular melanoma, which method comprises
administering to the patient an effective amount of one or more of
the agents selected from the MCSP peptides and/or the fusion
proteins as defined in (1) and (2) above, the nucleic acids as
defined in (3) above, the vectors as defined in (4) above, the full
length MCSP protein of SEQ ID NO:1 and fragments thereof, nucleic
acid sequences encoding the full length MCSP protein of SEQ ID NO:1
or fragments thereof, and vectors comprising nucleic acid sequences
encoding the full length MCSP protein of SEQ ID NO:1 or fragments
thereof, the transfected/transformed cells as defined in (5) above,
the loaded mature DCs as defined in (9) above, the T-cell clones as
defined in (10) above, the antibodies as defined in (11) above,
and/or the pharmaceutical composition as defined in (12) above; and
(18) a method for preparing an MCSP peptide or a fusion protein as
defined in (1) and (2) above, which method comprises culturing a
cell as defined in (5) above and isolating the expressed peptide or
fusion protein, or comprises chemical synthesis (i.e. solid phase)
of the MCSP peptides or fusion proteins.
BRIEF DESCRIPTION OF THE FIGURES
[0028] FIG. 1: Experimental protocol for activation of anti-MCSP
CD4.sup.+ T-cells. Peripheral blood mononuclear cells (PBMCs) were
isolated from a blood sample drawn with consent from a healthy
donor. Using magnetic cell sorting (MACS) technology the CD4.sup.+
T-cells were isolated from the PBMCs, and seeded at 10.sup.5 cells
per well of a microtiter plate. The CD4.sup.+ T-cells depleted
fraction of the PBMCs was briefly cultured, in order to obtain
adherent cells. Non-adherent cells were discarded, and the adherent
cells were then exposed to a differentiation cocktail comprising
GM-CSF and IL-4, and cultured for further 5 to 7 days, in order to
obtain immature DCs, identifiable by their phenotype. The immature
DCs were contacted for 1 h with 10 .mu.g/ml of the candidate
peptide derived from MCSP (SQVLFSVTRGAHYGEL (SEQ ID NO:12),
VRYLSTDPQHHAYDTV (SEQ ID NO:13), GEALVNFTQAEVYAGN (SEQ ID NO:14))
or as a control peptide the MAGE-3.DP4 epitope, in order to obtain
loaded DCs functioning as APCs. The ability of these APCs to induce
in vitro activation and proliferation of specific CD4.sup.+ T-cells
was tested by adding 10.sup.4 loaded DCs to each well of the
microtiter plate already containing CD4.sup.+ T-cells. The mixed
CD4.sup.+ T-cell/DCs were cultured on day 0 in the presence of
IL-6, IL-12 and TNF-.alpha. and weekly restimulated with DCs
freshly pulsed/loaded with the peptides and addition of IL-2 and
IL-7. The CD4.sup.+ T-cell comprising microcultures were assessed
on day 30 for their capacity to produce IFN-.gamma. when stimulated
with autologous target cells (Epstein Barr virus (EBV)-transformed
B cells (EBV-B cells)) loaded with the relevant MCSP peptide or the
control peptide, using an IFN-.gamma. ELISA.
[0029] FIG. 2: Experimental protocol for activation of anti-MCSP
CD4.sup.+ T-cells. Peripheral blood mononuclear cells (PBMCs) were
isolated from a blood sample drawn with consent from a healthy
donor. Using magnetic cell sorting (MACS) technology the CD4.sup.+
T-cells were isolated from the PBMCs, and seeded at 10.sup.5 cells
per well of a microtiter plate. The CD4.sup.+ T-cells depleted
fraction of the PBMCs was briefly cultured, in order to obtain
adherent cells. Non-adherent cells were discarded, and the adherent
cells were then exposed to a differentiation cocktail comprising
GM-CSF and IL-4, and cultured for further 5 to 7 days, in order to
obtain immature DCs, identifiable by their phenotype. The immature
DCs were contacted overnight with 50 .mu.g/ml of a 42 mer peptide
derived from MCSP (MCSP-peptide.sub.673-714) having the sequence
LAQGSAMPILPANLSVETNAVGQDVSVLFRVTGALQFGELQK (SEQ ID NO:3).
[0030] The contact period was longer to allow for processing of the
42 mer and presentation of peptides derived there from.
Furthermore, 6 h after exposure to the peptide, a cytokine
maturation cocktail comprising IL-1.beta., IL-6, TNF-.alpha. and
PGE.sub.2 was added to the DCs, to induce maturation. The ability
of these DCs to function as APCs capable of inducing in vitro
activation and proliferation of specific CD4.sup.+ T-cells, was
tested by adding 10.sup.4 loaded DCs to each well of the microtiter
already containing CD4.sup.+ T-cells. The mixed CD4.sup.+
T-cell/DCs were cultured on day 0 in the presence of IL-6, IL-12
and TNF-.alpha. and weekly restimulated with DCs freshly
pulsed/loaded with the peptide and addition of IL-2 and IL-7. The
CD4.sup.+ T-cell comprising microcultures were assessed on day 30
for their capacity to produce IFN-.gamma. when stimulated with
autologous target cells (Epstein Barr virus (EBV)-transformed B
cells (EBV-B cells)) loaded with the MCSP peptide or an irrelevant
control peptide, using an IFN-.gamma. ELISA. Three peptide-specific
CD4.sup.+ T-cell-clones were obtained, and the following
experiments were conducted with clone C2/25 (clone 25).
[0031] FIG. 3: Autologous EBV-B-cells of donor 4800 were
pulsed/loaded overnight with the 42 mer MCSP-peptide.sub.673-714 (5
.mu.M) or a control peptide, washed and used as stimulator cells.
4.times.10.sup.3 CD4.sup.+ T-cells were co-incubated with
1.5.times.10.sup.4 stimulator cells for 20 h, before the
IFN-.gamma. concentration in the supernatant was measured by an
ELISA. Values represent means of triplicates.
[0032] FIG. 4: Autologous EBV-B-cells (1.5.times.10.sup.4) were
pulsed/loaded with overlapping peptide fragments of the 42 mer
MCSP-peptide.sub.673-714 (1 .mu.M) for 1 h, washed and co-cultured
for 20 h with the CD4.sup.+ T-cell-clone 25 in order to test for
recognition and IFN-.gamma. secretion. An IFN-.gamma. ELISA was
performed.
[0033] FIG. 5: Fine-specificity of the shortest MCSP epitope
recognized by CD4.sup.+ T-cell-clone 25. Autologous EBV-B cells
(1.5.times.10.sup.4) were pulsed for 1 h with a panel of truncated
peptides (1 .mu.M) and tested for recognition by the CD4.sup.+ T
cell clone. IFN-.gamma. production was measured after overnight
co-culture by ELISA. The set of truncated peptides was derived from
the overlapping peptide VGQDVSVLFRVTGALQ (SEQ ID NO:9). The
peptides were truncated for up to 7 amino acids at the N- or
C-terminus. Truncations for up to 3 amino acids at both N- and
C-terminus were tolerated. Truncation going beyond the D at amino
acid position 696 (SEQ ID NO:1) or the A at amino acid position
706, at the N terminus or C-terminus, respectively, resulted in the
loss of recognition by the T cell clone, as measured by an
IFN-.gamma. ELISA.
[0034] FIG. 6: Autologous EBV-B cells (1.5.times.10.sup.4) pulsed
with 1 .mu.M of the 16-mer MCSP peptide VGQDVSVLFRVTGALQ (SEQ ID
NO:9) were used as stimulator cells in the presence of different
blocking antibodies (anti-DR, anti-DQ or anti-DP). Also a control
with no antibody (no Ab) was set up. All antibodies were used at a
final concentration of 5 .mu.g/ml each. IFN-.gamma. production of
the CD4.sup.+ T cells was measured after overnight co-culture (20
h) by ELISA.
[0035] FIG. 7: 1.5.times.10.sup.4 cells of several EBV-B cell lines
(LP2-, LB1981-, R12-, PV6-, AC 42- and 4800-EBV) with different HLA
class II molecules (HLA-DR11 positive or HLA-DR11 negative) were
pulsed/loaded for 1 h with MCSP peptide VGQDVSVLFRVTGALQ (SEQ ID
NO:9) (1 .mu.M, white bar) and tested for recognition by the
CD4.sup.+ T cell clone. As control, the cells were pulsed in the
absence of protein (black spotted bar). IFN-.gamma. production of
the CD4.sup.+ T cells was measured after overnight co-culture (20
h) by ELISA.
[0036] FIG. 8: EBV-B cell lines HLA-DR11 positive (4800- and MVGS
EBV) or not (MMDH EBV) were transduced with a retrovirus coding for
an Ii.-MCSP fusion protein (spotted bars) or an Ii.-MAGE-3 protein
(white bars) as a control The fusion protein comprised amino acid
residues 392 to 748 of the MCSP. Clone 25 was then co-cultured with
1.5.times.10.sup.4 stimulator cells for 20 h. IFN-.gamma.
production of the CD4.sup.+ T cells was measured after overnight
co-culture by ELISA.
[0037] FIG. 9: Clone 25 was stimulated by HLA-matched or
-mismatched MCSP-expressing melanoma cell lines (2.times.10.sup.4).
IFN-.gamma. production of the CD4.sup.+ T cells was measured after
overnight co-culture by ELISA.
[0038] FIG. 10: CD4.sup.+ T cells (100,000 per 96 round-bottomed
microwell) of donor 11325 were stimulated with autologous
monocyte-derived dendritic cells (10,000 per well) loaded overnight
with the long TAT-MCSP-peptide (SEQ ID NO:54; 10 .mu.M). After 3
weekly re-stimulations, microcultures were tested for their
IFN-.gamma. production when stimulated with autologous EBV-B cells
loaded with the MCSP peptide or a control peptide.
[0039] FIG. 11: Autologous EBV-B cells of donor 11325 were loaded
overnight with the long TAT-MCSP-peptide (SEQ ID NO:54; 10 .mu.M)
or a control peptide, washed and used as stimulator cells. 4,000
CD4.sup.+ T-cells were co-incubated with 15,000 stimulator cells
and after 20 h the IFN-.gamma. concentration in the supernatant was
measured by ELISA.
[0040] FIG. 12: Autologous EBV-B cells (15,000) were pulsed with
overlapping truncated peptides (5 .mu.g/ml) for 1 h or in the case
of the long TAT-MCSP-peptide (SEQ ID NO:54) overnight at 10 .mu.M,
washed and tested for recognition by the CD4.sup.+ T-cell clone
(4,000 cells per well) after 20 h co-culture by IFN-.gamma.-ELISA.
Values shown are the mean of duplicate determinations, bars,
SD.
[0041] FIG. 13: Autologous EBV-B cells (15,000) were pulsed for 1 h
with a panel of truncated peptides (5 .mu.g/ml) and tested for
recognition by the CD4.sup.+ T cell clone (4,000 cells per well).
IFN-.gamma. production was measured after overnight co-culture by
ELISA. Values shown are the mean of duplicate determinations, bars,
SD.
[0042] FIG. 14: Autologous EBV-B cells (15,000) pulsed with 16-mer
MCSP peptide (aa 1281-1296 (SEQ ID NO:48), 5 .mu.g/ml) were used as
stimulator cells in the presence of different blocking antibodies.
All antibodies were used at a final concentration of 5 .mu.g/ml
each. IFN-.gamma. production by CD4.sup.+ T cells (4,000 cells per
well) was measured after overnight co-culture by ELISA.
[0043] FIG. 15: Several EBV-B cell lines with different HLA class
II molecules were pulsed with peptide MCSP.sub.1281-1296 (SEQ ID
NO:48; 5 .mu.g/ml) and tested for recognition (at 15,000 cells per
well) by the CD4.sup.+ T cell clone (4,000 cells per well).
IFN-.gamma. production by CD4.sup.+ T cells was measured after
overnight co-culture by ELISA.
[0044] FIG. 16: Clone 3 was stimulated by HLA-matched (ER-MEL-4) or
-mismatched MCSP-expressing melanoma cell lines (20,000) which had
been thawed 48 h before to allow the formation of a confluent cell
layer. IFN-.gamma. production by CD4.sup.+ T cells (4,000 cells per
well) was measured after overnight co-culture by ELISA.
[0045] FIG. 17: CD8.sup.+ T cells (150,000 per 96 round-bottomed
microwells) of donor 11325 were stimulated with autologous
monocyte-derived dendritic cells (15,000 per well) loaded overnight
with the long TAT-MCSP-peptide (SEQ ID NO:54; 10 .mu.M). After 3
weekly restimulations microcultures were tested for their
IFN-.gamma. production when stimulated with autologous EBV-B cells
loaded with the TAT-MCSP peptide (SEQ ID NO:54) or a control
peptide.
[0046] FIG. 18: Autologous EBV-B-cells of donor 11325 were loaded
overnight with the long TAT-MCSP-peptide (SEQ ID NO:54; 10 .mu.M)
or a control peptide, washed and used as stimulator cells. Aliquots
of approximately 4,000 CD8.sup.+ T-cells from each 96-well
microculture were co-incubated with 15,000 stimulator cells and
after 20 h the IFN-.gamma. concentration in the supernatant was
measured by ELISA. Values shown represent duplicate determinations
from two representative positive tested microcultures out of 96
tested. Bars represent standard deviations.
DETAILED DESCRIPTION OF THE INVENTION
[0047] The aim of the present invention was the identification of
T-cell epitopes from the melanoma-associated chondroitin sulfate
proteoglycan (MCSP), a tumour antigen with potential benefit for
vaccination and immunomonitoring of cancer, especially of melanoma
patients. The identification of tumour antigens recognized by
cytolytic CD8.sup.+ T cells (CTLs) on human tumour cells has opened
new avenues in cancer immunotherapy. There is consensus that the
induction of both tumour-specific CTLs and CD4.sup.+ T helper cells
is necessary for an optimal antitumour immunity. Unfortunately,
only a few tumour-specific helper T cell epitopes have been
described so far. Therefore the present invention focuses on the
identification of melanoma antigens recognized by CD4.sup.+ T
cells. One interesting candidate antigen is the human
melanoma-associated chondroitin sulfate proteoglycan (MCSP), which
is expressed on >90% of human melanoma tissues and induces
strong humoral responses in mice. The demonstration of humoral
anti-MCSP immunity in a mouse and a human model implicated the
co-existence of MCSP-specific CD4.sup.+ T-cells, therefore the
present invention is focused on the identification and verification
of MCSP-specific CD4.sup.+ T-cell responses. In addition, the
presence of MCSP-specific CD8.sup.+ T-cell responses is
demonstrated.
DEFINITIONS
[0048] A "T cell" in the context of present invention is a CD3+
lymphocyte. Preferably, the T cells of present invention are
CD4.sup.+ or CD8.sup.+ cells, more preferably CD4.sup.+ cells.
[0049] The expression "antigenic T-cell stimulatory peptide" refers
to the ability of a peptide to be recognized by a T-cell when bound
to a given HLA molecule and to stimulate the T-cell to secrete
cytokines and/or to proliferate and/or to display lytic activity.
The expression "functional variant" refers to the antigenic T-cell
stimulatory peptide, comprising at least one amino acid addition or
substitution not affecting the ability of the peptide to stimulate
a T-cell. Preferably an addition arises from adding between 1 to 15
amino acids, preferably 1 to 10, most preferably 5 to 10 amino
acids anywhere to the 100 amino acid core peptide comprising
sequence at amino acid position 644 to 743 (SEQ ID NO:1), or from
adding between 1 to 15, preferably 1 to 10, most preferably 5 to 10
amino acids to the N- and/or C-terminus of the peptide fragments
derived from the amino acid region 673-714 of SEQ ID NO:1. The term
"substitution" refers to the replacement of an amino acid with an
homologous amino acid, which are known to the person skilled in the
art. A homologous substitution is also referred to as "conservative
amino acid change". Furthermore, it also refers to the replacement
of an amino acid with the corresponding D-amino acid, or a
derivatized amino acid. A "derivatized amino acid" is an amino acid
which comprises a modified functional group, such as a free amino
group which has been chemically modified to form amine
hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,
t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
Free carboxyl groups may be derivatized to form salts, methyl and
ethyl esters or other types of esters or hydrazides. Free hydroxyl
groups may be derivatized to form O-acyl or O-alkyl derivatives.
The imidazol nitrogen of histidine may be derivatized to form
N-imbenzylhistidine. Also included are naturally-occurring amino
acid derivatives of the twenty standard amino acids. For example:
4-hydroxyproline may be substituted for proline, 5-hydroxylysine
may be substituted for lysine, homoserine may be substituted for
serine, 3-methylhistidine may be substituted for histidine, and
ornithine or citrulline may be substituted for lysine. The
functional variant may comprise up to 15 substitutions, preferably
1 to 10, most preferably 1 to 5 substitutions.
[0050] "Fusion peptide" according to the invention refers to
peptides where a first domain comprising an antigenic T cell
stimulating peptide (i.e. the MCSP domain) is directly or through a
linker peptide fused to a second function protein or peptide
donor.
[0051] The term "one or more of the MCSP peptides and fusion
proteins" includes the single MCSP peptide or fusion protein and
also mixtures out of said MCSP peptides and/or fusion proteins of
the invention. Where applicable, the term "one or more" is to be
construed similarly for the nucleic acids and vectors, for the
cells and for the antibodies of the invention.
[0052] The expression "endosomal targeting signal" refers to a
short peptide sequence, in general comprising up to 30 amino acids,
which directs the protein and/or peptide with which its
functionally linked into the endosome. "Functionally linked" is
here to be understood to refer to a peptide bond between the
sequence comprising the endosomal targeting signal sequence and the
peptide according to the invention. A short peptide linker may lie
between the endosomal targeting sequence and the peptide/protein
sequence to be targeted into the endosome.
[0053] The expression "nucleic acid" refers to any nucleic acid
known to a person skilled in the art, including DNA and RNA,
whereby the nucleic acid may be double-stranded, single-stranded,
circular and/or linear. It also includes nucleic acid molecules
with modifications to their bases as well as the sugar-phosphate
backbone.
[0054] The term "vector" is generally understood by a person
skilled in the art and comprises a DNA molecule, or its
corresponding RNA molecule, derived from a plasmid, a bacterial
phage, or a mammalian or insect virus, into which fragments of DNA
may be inserted or cloned. A vector comprises one or more unique
restriction enzyme sites and may be capable of autonomous
replication. Furthermore the vector is capable of expressing the
inserted or cloned fragment by providing transcription control
elements, such as a promoter and transcription termination signals.
The DNA fragment inserted in to the vector, here the DNA coding for
the peptide according to the invention, is functionally linked to
said transcription control elements. In addition the vector may
also comprise a nucleic acid sequence encoding HLA-DR11. Such a
vector, when transfected into a cell, gives rise to an artificial
antigen presenting cell. Examples of suitable vectors include
retroviral vectors, vaccinia vectors, adenoviral vectors, herpes
virus vectors, fowl pox virus vectors, plasmids, baculovirus
transfer vectors.
[0055] The terms "transfected" and "transformed" refer to the
introduction of a nucleic acid into a cell. Various transfection
and transformation methods are known to the person skilled in the
art. The term "transfection" generally describes the introduction
of a nucleic acid into a mammalian cell, whereas the term
"transformation" describes the uptake of a nucleic acid by a
microbial cell such as fungal cells or prokaryotes. The means by
which the nucleic acids are introduced into the cell include
microinjection, lipofection, electroporation, calcium phosphate
transfection, DEAE-dextran transfection or infection with a
recombinant virus harbouring said nucleic acid (Sambrook et al. in
"Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Press,
Plainview, N.Y. (1989)).
[0056] A CD4.sup.+ T-cell clone is said to be "specific" for a
peptide, if upon exposure to the peptide bound to a HLA-class II
molecule, the CD4.sup.+ T-cell recognizes the HLA-peptide-complex
with its T cell receptor resulting in cytokine secretion by the
CD4.sup.+ T-cell, in particular TNF-.alpha. and/or IFN-.gamma.
secretion, and/or other cytokines, as well as proliferation of the
CD4.sup.+ T-cell.
[0057] Analogously, a CD8.sup.+ T cell clone is said to be
"specific" for a peptide, if upon exposure to the peptide bound to
a HLA-class II molecule, the CD8.sup.+ T-cell recognizes the
HLA-peptide-complex with its T cell receptor resulting in cytokine
secretion by the CD8.sup.+ T-cell, in particular TNF-.alpha. and/or
IFN-.gamma. secretion, and/or other cytokines, as well as
proliferation of the CD8.sup.+ T-cell.
[0058] The term "antigen presenting cell" (APC) is generally
understood by the person skilled in the art. It refers to highly
specialized cells that can process antigens and display their
peptide fragments on the cell surface together with molecules
required for lymphocyte activation. The main APCs for T cells are
DCs, macrophages, and B-cells, while the main APCs for B-cells are
follicular DCs. In the sense of the present invention the term APC
also comprises artificial APCs, which can be generated by
co-expressing the nucleic acids encoding the molecules required for
lymphocyte activation, in particular HLA-class II molecules, such
as HLA-DR11, as well as the nucleic acids encoding the peptides
according to the invention, in cells not normally functioning as
APCs. The co-expression may be achieved via transfection with a
single vector comprising the coding sequences for both, the peptide
as well as the HLA-class II molecule, e.g. HLA-DR11; or by
co-transfection of two individual vector molecules, one encoding
anyone of the peptides according to the invention and the other one
the HLA-class II molecule, e.g. HLA-DR11.
[0059] The expression "stimulator cell" refers to cells used in
assays to test the ability of T-cells to respond to the antigenic
peptide according to the invention. In general stimulator cells are
characterized by the expression of antigenic peptides bound to the
groove of HLA class I/II molecule and the expression of
costimulatory molecules such as CD80 and CD86. Stimulator cells are
generally selected from the group of Epstein-Barr virus (EBV)
transformed autologous or allogenic B cells (in short: EBV-B cells)
pulsed with the peptide according to the invention. Autologous
cells are preferred. Due to the transformation with EBV the B cells
are immortalized. Stimulator cells can also be selected from the
group of macrophages, PBMCS, DCs, and CD40-ligand stimulated
B-cells. The expression "pulsed", "pulsed with", "loaded", "loaded
with", "pulsed/loaded" or "pulsed/loaded with" refers to cells
displaying anyone of the peptides according to the invention. The
pulsing/loading is achieved by exposing cells, e.g. DCs or EBV-B
cells, to said peptide for an amount of time sufficient to allow
uptake. In general, 1 h is sufficient to achieve uptaking and
displaying, but the exposure time may be expanded for up to 20
h.
[0060] In the context of the present invention the expression
"assessing the ability of proliferating T-cells"--notably the
CD4.sup.+ and CD8.sup.+ T cells--"to produce TNF-.alpha. and/or
IFN-.gamma." is meant to refer to various assays allowing
measurement of said cytokines. To the person skilled in the art it
is clear that other cytokines, for example GM-CSF or IL-2, can be
assessed in analogous ways. In particular, the "assessing" can be
achieved through a cytokine-specific ELISA assay, but also through
other methods, such as bioassays, in which cells responsive to the
secreted cytokine are tested for responsiveness (e.g.
proliferation) in the presence of a test sample or the ELISPOT
assay, cytokine bead arrays, quantitative real-time PCR for
cytokines or intracytoplasmatic cytokine staining.
[0061] The term "feeder cells" refers to cells which may secrete
protein factors or give other types of stimulatory signals
supporting the proliferation of T cells. Feeder cells may be
selected from the group of immortalized B-cells, such as allogenic
as well as autologous B-cells, PBMCs and CD40-ligand activated
B-cells. Preferably the feeder cells are LG2-EBV cells.
[0062] The term "cloning . . . the T-cell" refers to obtaining a
T-cell population derived from a single T-cell, whereby all cells
of the population have an identical genotype and phenotype. Cloning
is achieved by limiting dilution culture, i.e. harvesting a T-cell
population determined to secrete TNF-.alpha., IFN-.gamma. and/or
other cytokines, e.g. GM-CSF and diluting the harvested T-cell
population by a factor 10.sup.1 to 10.sup.8, plating out and
co-culturing said diluted T-cell populations in the presence of
feeder cells. Individual T-cell clones can be obtained in this
way.
[0063] The term "antibody" is generally understood by the person
skilled in the art. In particular it refers to proteins that bind
specifically to particular antigens, in the context of the present
invention to those which bind to the peptides according to the
invention, and are produced in response to immunization with the
antigen. They bind to and neutralize cells displaying the antigen
and prepare them for uptake and destruction by phagocytes. The term
"protective antibody" refers to an antibody which protects an
organism from harmful matter, including tumour cells expressing and
displaying an antigenic peptide according to the invention. As used
herein, the term "antibody" refers to polyclonal antibodies,
monoclonal antibodies, humanized antibodies, single-chain
antibodies, and fragments thereof such as Fab, F(ab')2, Fv, and
other fragments which retain the antigen binding function and
specificity of the parent antibody. The term "monoclonal antibody"
refers to an antibody composition having a homogeneous antibody
population. The term is not limited regarding the species or source
of the antibody, nor is it intended to be limited by the manner in
which it is made. The term encompasses whole immunoglobulins as
well as fragments such as Fab, F(ab')2, Fv, and others which retain
the antigen binding function and specificity of the antibody.
[0064] As used herein, the term "human antibodies" means that the
framework regions of an immunoglobulin are derived from human
immunoglobulin sequences. As used herein, the term "single chain
antibody fragments" (scFv) refer to antibodies prepared by
determining the binding domains (both heavy and light chains) of a
binding antibody, and supplying a linking moiety which permits
preservation of the binding function. This form, in essence, is a
radically abbreviated antibody, having only that part of the
variable domain necessary for binding to the antigen. Determination
and construction of single chain antibodies are described in U.S.
Pat. No. 4,946,778 by Ladner et al.
[0065] The expression "pharmaceutically acceptable carrier" refers
to all known substances used for the formulation of a medicament,
not themselves being an active ingredient of the medicament. The
expression "diagnostically acceptable carrier refers to substances
used for the formulation of diagnostika which are not interfering
with the reaction indicative for the diagnostically targeted
disease.
[0066] The term "vaccine" refers to a composition either used
prophylactically or therapeutically to prevent or treat diseases
associated with the expression of the peptides according to the
invention or with the expression of MCSP itself. In particular the
vaccine of present invention is used against melanoma and other
MCSP expressing tumours such as breast cancer, notably lobular
breast carcinoma, astrocytoma, glioma, glioblastoma, neuroblastoma,
sarcoma and certain types of leukaemia. The vaccine is
characterized in that it triggers an immune response, in particular
a cellular immune response. As adjuvant the vaccine may comprise
Freund's complete adjuvants, Freund's incomplete adjuvants,
Montanide ISA Adjuvants (Seppic, Paris, France), Ribi's Adjuvants
(Ribi ImmunoChem Research, Inc., Hamilton, Mont.), Hunter's
TiterMax (CytRx Corp., Norcross, Ga.), Aluminum Salt Adjuvants,
Gerbu Adjuvant (Gerbu Biotechnik GmbH, Gaiberg, Germany/C-C
Biotech, Poway, Calif.), MPL (Glaxo Smithkline), AS02B (Glaxo
Smithkline), QS21 (Glaxo Smithkline) and/or Toll like receptor
agonists such as imiquimod (3M Medica, Neuss, Germany).
[0067] The expression "immune cell" refers to any cell
participating in the immune response. In particular it refers to
B-cells, T-cells, monocytes, macrophages, dendritic cells, NK-cells
and/or NKT-cells.
[0068] The term "melanoma" refers to all kinds of melanoma
including cutaneous melanoma, ocular melanoma, metastatic melanoma,
melanomas derived from either melanocytes or melanocyte related
nevus cells, melanocarcinoma, melanoepitheliomas, melanosarcomas,
melanoma in situ, superficial spreading melanoma, nodular melanoma,
lentigo maligna melanoma, acral lentiginous melanoma, invasive
melanoma or familial atypical mole and melanoma (FAM-M) syndrome.
Such melanomas in mammals may be caused by chromosomal
abnormalities, degenerative growth and developmental disorders,
mitogenic agents, ultraviolet radiation (UV), viral infections,
inappropriate tissue expression of a gene, alterations in
expression of a gene, and presentation on a cell, or carcinogenic
agents.
[0069] The term "certain kinds of leukaemia" refers to adult acute
lymphoblastic leukaemia (ALL) and childhood acute myeloid leukaemia
(AML).
[0070] The expression "diagnostic marker for cancer" refers to
molecules specifically found on cancerous growth. These may be
proteins and peptides newly expressed, mutated, or aberrantly
expressed in tumour cells. They allow to distinguish the tumour
cell from a healthy cell, which does not express said proteins or
peptides.
[0071] The term "medicament" in the context of the present
invention refers to a vaccine, a diagnostic agent as well as to any
other therapeutically active pharmaceutical composition.
[0072] The term "immunomonitoring" refers to a diagnostic
monitoring procedure, whereby cells of the immune system capable of
binding to the peptide of the invention, such as B-cells or T-cells
are quantified. High numbers of these cells specific for anyone of
the peptides according to the invention are likely to be diagnostic
of a relevant disease, such as a tumour, in particular melanoma, or
an indication that these cells are involved in immunity to the
disease. In particular useful for immunomonitoring may be multimers
(dimers, trimers, tetramers, pentamers, hexamers or oligomers) of a
class II HLA molecule comprising a covalently or non-covalently
bound peptide according to the invention, which is conjugated with
a detectable label. A label may be selected from the group of
fluorescent moieties, radionuclides, or enzymes that catalyze a
reaction resulting in a product that absorbs or emits light of a
defined wavelength. Such a multimer may be used to quantify in
vitro T cells or B-cells from a subject, e.g. a human patient,
bearing cell surface receptors that are specific for, and therefore
will bind such multimers.
[0073] The expression "biological sample" refers to any material
isolated from a subject, e.g. a human patient. In particular it
refers to biopsy material and/or a blood sample obtained from a
subject.
[0074] The term "fragment of the full length MCSP protein" in the
context of present invention designates a fragment of the full
length amino acid sequence of MCSP as represented by SEQ ID NO:1
which is immunogenic, i.e. which is able to stimulate an immune
response in mammals, preferably in humans. Said fragment contains a
chain of consecutive amino acids of said SEQ ID NO:1, preferably at
least 10 consecutive amino acids, more preferably at least 13
consecutive amino acids. Its maximum length is 100 amino acids,
more preferably 42 amino acids.
[0075] The present invention discloses an antigenic T-cell
stimulatory peptide from the melanoma-associated chondroitin
sulfate proteoglycan (MCSP) (SEQ ID NO:1) of 100 amino acids in
length comprising amino acids 644 to 743 of MCSP (SEQ ID NO:1),
and/or a fragment thereof of at least 8 amino acids, and/or a
functional variant thereof comprising one or more amino acid
additions or substitutions. It has to be noted that the sequence
given as SEQ ID NO:1 corresponds to the native protein, i.e.
comprises the 29 amino acid signal peptide of the MCSP. The nucleic
acid encoding the MCSP as given in SEQ ID NO:1 is shown in SEQ ID
NO:2.
[0076] The MCSP peptide of embodiment (1) comprises at least 10,
preferably at least 12, more preferably at least 13 amino acid
residues. In one preferred aspect, the peptide of embodiment (1)
comprises the MCSP fragment represented by amino acid residues 695
to 705 of SEQ ID NO:1 (QDVSVLFRVTG) or by amino acid residues 1285
to 1295 of SEQ ID NO:1 (GYLVMVSRGAL). In an even more preferred
aspect, it comprises
(i) at least 13 consecutive amino acid residues out of the MCSP
segment represented by amino acid residues 644 to 743 of SEQ ID
NO:1, preferably comprises the amino acid residues 695 to 705 of
SEQ ID NO:1; or (ii) at least 12 consecutive amino acid residues
out of the MCSP segment represented by amino acid residues 1270 to
1300 of SEQ ID NO:1, preferably comprises the amino acid residues
1285 to 1295 of SEQ ID NO:1.
[0077] In particular the present invention relates to a peptide
fragment of 13 to 42 amino acids in length, which is derived from
the 100 amino acid region at amino acid position 644 to 743 of MCSP
(SEQ ID NO:1). The peptide of embodiment (1) is preferably derived
from the MCSP fragment represented by amino acid residues 673 to
714 or 1270 to 1300 of SEQ ID NO: 1. Preferred peptide fragments
comprise a sequence selected from the following amino acid
sequences:
TABLE-US-00001 (SEQ ID NO:3)
LAQGSAMPILPANLSVETNAVGQDVSVLFRVTGALQFGELQK, (SEQ ID NO:4)
LAQGSAMPILPANLSV; (SEQ ID NO:5) SAMPILPANLSVETNA; (SEQ ID NO:6)
ILPANLSVETNAVGQD; (SEQ ID NO:7) NLSVETNAVGQDVSVL; (SEQ ID NO:8)
ETNAVGQDVSVLFRVT; (SEQ ID NO:9) VGQDVSVLFRVTGALQ; (SEQ ID NO:10)
VSVLFRVTGALQFGEL; or (SEQ ID NO:11) FRVTGALQFGELQK. Especially
preferred peptides have a sequence selected from (SEQ ID NO: 3)
LAQGSAMPILPANLSVETNAVGQDVSVLFRVTGALQFGELQK, (SEQ ID NO:8)
ETNAVGQDVSVLFRVT and (SEQ ID NO:9) VGQDVSVLFRVTGALQ,
[0078] Said peptide fragments may be shortened by up to three
C-terminal and/or N-terminal amino acids, without loosing their
antigenic T-cell stimulatory function. Preferably, said peptide
fragments are not shortened on their termini.
[0079] Moreover in particular the present invention relates to the
peptide fragment of 12 to 30 amino acids in length, derived from
the 31 amino acid region at amino acid position 1270 to 1300 of
MCSP (SEQ ID NO:1). Preferred peptide fragments comprise a sequence
selected from the following amino acid sequences:
TABLE-US-00002 (SEQ ID NO:36) PPADIVFSVKSPPSAGYLVMVSRGALADEPP, (SEQ
ID NO:37) PPSAGYLVMVS, (SEQ ID NO:38) PPSAGYLVMVSR, (SEQ ID NO:39)
PPSAGYLVMVSRG, (SEQ ID NO:40) PPSAGYLVMVSRGA, (SEQ ID NO:41)
PPSAGYLVMVSRGAL, (SEQ ID NO:42) PPSAGYLVMVSRGALA, (SEQ ID NO:43)
PSAGYLVMVSRGALA, (SEQ ID NO:44) SAGYLVMVSRGALA, (SEQ ID NO:45)
AGYLVMVSRGALA, (SEQ ID NO:46) GYLVMVSRGALA, (SEQ ID NO:47)
YLVMVSRGALA, (SEQ ID NO:48) PPADIVFSVKSPPSAG, (SEQ ID NO:49)
IVFSVKSPPSAGYLV, (SEQ ID NO:50) SVKSPPSAGYLVMVSR, (SEQ ID NO:51)
GYLVMVSRGALADEPP.
[0080] Especially preferred peptides have a sequence selected
from
TABLE-US-00003 (SEQ ID NO:36) PPADIVFSVKSPPSAGYLVMVSRGALADEPP, (SEQ
ID NO:41) PPSAGYLVMVSRGAL, (SEQ ID NO:42) PPSAGYLVMVSRGALA, (SEQ ID
NO:43) PSAGYLVMVSRGALA, (SEQ ID NO:44) SAGYLVMVSRGALA, (SEQ ID
NO:45) AGYLVMVSRGALA, (SEQ ID NO:46) GYLVMVSRGALA, and (SEQ ID
NO:51) GYLVMVSRGALADEPP.
[0081] Said peptide fragments may be shortened by up to three
C-terminal and/or N-terminal amino acids. Preferably, said peptide
fragments are not shortened on their termini.
[0082] Anyone of the above described peptides and/or peptide
fragments and/or functional variant thereof, can be found either in
full-length or in fragmented form associated with HLA-DR molecules,
e.g. with HLA-DR-11, i.e. anyone of these peptides can be presented
by HLA-DR11. Said peptides may also be found associated with HLA-DP
molecules.
[0083] In a further embodiment the above described peptides
comprise at least one conservative amino acid exchange, at least
one amino acid replaced by a D-amino acid, and/or at least one
amide bond selected from the group of psi[CH.sub.2NH]-reduced amide
peptide bonds, psi[COCH.sub.2]-ketomethylene peptide bond,
psi[C(CN)NH]-(cyanomethylene) amino peptide bond, a
psi[CH.sub.2CH(OH)]-hydroxyethylene peptide bond, a
psi[CH.sub.2O)]-peptide bond and psi[CH.sub.2S]-thiomethylene
peptide bond. Peptides comprising any such modification are more
stable in comparison to unmodified peptides. In particular they
have an extended plasma half life. This is of particular advantage
because it allows for administration of lower dosages of a
medicament comprising said peptides.
[0084] Anyone of the above described MCSP peptides may be part of
the fusion protein according to embodiment (2), i.e. may form the
MCSP domain of said fusion protein. Said fusion protein preferably
comprises one second domain, but it is to be understood that it may
also comprise more than one second domains which may be same or
different and selected, e.g., from those domains mentioned
below.
[0085] The at least one second domain of the fusion protein, i.e.
the fusion partner(s) of the MCSP domain, preferably is a protein
and/or peptide and comprises an endosomal targeting signal, and/or
is selected from the group of the human invariable chain (Ii), a
peptide fragment thereof comprising amino acid residues 1-80, the
lysosome-associated membrane protein (LAMP-1) and DC-LAMP. Thus the
fusion protein comprises at least two domains, viz. the MCSP domain
comprising anyone of the MCSP peptides according to the invention,
and the second domain comprising a protein or peptide with an
endosomal targeting signal. The order of the domains will be chosen
to allow for functionality of the domains, i.e; the endosomal
targeting signal must be capable of directing the fusion protein
into the endosome.
[0086] The first and the at least one second domain of the fusion
protein may be connected directly (i.e. by a peptide bond) or
through a linker peptide. Suitable linker peptides have a length of
1 to 20, preferably of 1 to 12 amino acid residues and are
preferably comprised of flexible hydrophobic amino acid residues.
Particularly useful linkers are polypeptides such as poly-Gly,
poly-Gly-Ser and the like.
[0087] In a further preferred embodiment the at least one second
domain of the fusion protein (2) is a protein transduction domain,
including protein transduction domains of the HIV TAT protein or
variants thereof. Preferred protein transduction domains within the
present invention are variants of the HIV TAT protein, particularly
preferred is the HIV TAT protein variant YARAAARQARA (SEQ ID NO:53)
linked to the N-terminus of the MCSP domain.
[0088] A particularly preferred fusion protein is a 42-mer having
the sequence YARAAARQARAPPADIVFSVKSPPSAGYLVMVSRGALADEPP (SEQ ID
NO:54).
[0089] In a further embodiment the present invention provides a
nucleic acid sequence encoding anyone of the MCSP peptides and/or
peptide fragments and/or functional variants and/or fusion proteins
described hereinbefore. A vector comprising any such nucleic acid
sequence is also part of the invention. An alternative embodiment
of said vector is a vector also comprising a nucleic acid sequence
encoding HLA-DR11. Such a vector enables the generation of
artificial APCs, by transfecting a cell normally not expressing the
HLA-DR11 complex with said vector. If a fibroblast is transfected
with said vector, the fibroblast will be turned into an artificial
APC. The cell, e.g. the fibroblast, transformed with said vector
will surface present the recombinant HLA-DR11 molecule as well as
the peptide encoded by said vector. The peptide will also occur
bound to the recombinant HLA-DR11 molecule.
[0090] A further embodiment of the invention is an isolated cell
transfected or transformed with anyone of the vector molecules
described above, and/or a cell comprising the nucleic acid molecule
according to the invention. Said cell is preferably selected from
the group of insect cells, plant cells, mammalian cells, preferably
a human cell or murine cell, most preferably primary cells such as
fibroblast, melanoma cells, DCs, B cells, macrophages, and
microorganism cells such as eukaryotic cells including fungal cells
(e.g. yeast cells, etc.) and prokaryotic cells, such as E.
coli.
[0091] Also an preferred aspect of embodiment (7) of the present
invention is a method to generate stable mature dendritic cells
(DCs) loaded with anyone of the MCSP peptides and fusion proteins
as described above, the full length MCSP protein of SEQ ID NO:1 and
fragments thereof. Preferably, said cells are loaded with one or
more of the MCSP peptides and fusion proteins as described above
and the full length MCSP protein, more preferably with the peptides
or fusion proteins as described above. The preferred method
comprises the following steps: (i) contacting isolated immature DCs
with anyone of the peptides or fusion proteins or the full length
protein (at concentrations of 0.01 to 1000 .mu.M) described above
to allow for uptake of said peptides or fusion proteins or full
length protein, or contacting them with anyone of the nucleic acid
molecules and/or the vector molecule described above, to allow for
uptake and subsequent expression of the peptide in the DC; and (ii)
maturing the contacted DCs by exposing them to a cytokine
comprising maturation cocktail (IL-1.beta., IL-6, PGE.sub.2 and
TNF-.alpha.) and/or monocyte conditioned medium. The contacting
phase may vary in time, but the immature DCs are brought in contact
with anyone of the peptides or fusion proteins according to the
invention for at least 1 h, however, the contact phase may be
extended for up to 30 h, whereby 20 h are preferred. The cytokine
comprising maturation cocktail is essentially composed of
IL-1.beta., IL-6, TNF-.alpha., and PGE.sub.2, and is added only if
a longer contact phase was chosen. Then it is added at about 6 h
past the initial contacting and left on the cells until the end of
the contact phase. For each cytokine a preferred range of
concentrations exists: IL-1.beta.: 1-20 ng/ml, preferably 1-10
ng/ml, most preferably 10 ng/ml; IL-6: 50-200 U/ml, preferably
80-150 U/ml, most preferably 100 U/ml; TNF-.alpha. 1-20 ng/ml,
preferably 1-10 ng/ml, most preferably 10 ng/ml; and PGE.sub.2;
0.1-10 ng/ml, preferably 0.1-5 ng/ml, most preferably 1 ng/ml.
[0092] It is to be understood that the mature DCs of the invention
generated in vitro are suitable for being administered
(retransfused) to a patient.
[0093] Also an preferred aspect of embodiment (8) of the present
invention is a method to generate a CD4.sup.+ T-cell clone specific
for anyone of the MCSP peptides described above, the fusion
proteins as described above, the full length MCSP protein of SEQ ID
NO:1 and fragments thereof. Preferably, said cells are specific for
anyone of the MCSP peptides or fusion proteins as described above
or the full length MCSP protein, more preferably for the peptides
or fusion proteins as described above. The preferred method
comprises the following steps: (i) contacting isolated CD4.sup.+
T-cells with an antigen presenting cell (APC) presenting anyone of
the peptides or proteins as described in the above paragraph,
whereby the APC is selected from the group of B-lymphocytes,
macrophages, and/or DCs. Preferentially the APC is a mature loaded
DC generated by the method of the invention described above; (ii)
co-culturing the isolated CD4.sup.+ T-cells with the APC for at
least 30 days, whereby freshly prepared APCs are added for at least
3 times to the original co-culture, and the CD4.sup.+ T-cells
proliferate; as culture medium is either RPMJ 1640 (Bio Whittaker)
used, supplemented with 10% heat inactivated human pool serum, 2 mM
L-glutamine and 20 .mu.g/ml gentamicin; alternatively J-VIVO15
(Cumber) is used, which is supplemented analogously; (iii)
assessing the ability of the proliferating CD4.sup.+ T-cells of
(ii) to produce TNF-alpha and/or IFN-.gamma. in response to the
addition of stimulator cells pulsed with anyone of the peptides
according to the invention, whereby the stimulator cells are
selected from the group of autologous or allogenic immortalized
B-cells, immortalized monocytes, macrophages or DCs, pulsed with
anyone of the peptides according to the invention; autologous cells
are preferred; (iv) cloning of the TNF-.alpha. and/or IFN-.gamma.
producing CD4.sup.+ T-cells of (iii) by limiting dilution culture
in the presence of autologous or allogenic (autologous are
preferred) stimulator cells pulsed with anyone of the peptides
according to the invention, and feeder cells, whereby the feeder
cells are selected from the group of allogenic or autologous
immortalized B-cells such as LG2-EBV; and (v) maintaining the
isolated CD4.sup.+ T-cell clone of step (iv) in the presence of
feeder cells in culture medium comprising interleukin-2 (IL-2),
interleukin-7 (IL-7) and phytohemagglutinin (PHA). The maintenance
medium is selected from the group of RPMJ 1640 (Bio Whittaker) and
J-VIVO 15 (Cumber) media, supplemented as described above with
human serum, L-glutamine and gentamicin, wherein IL-2, IL-7 and PHA
are present at concentrations ranging from 10 to 100 U/ml IL-2,
preferably 50 U/ml, 1 to 10 ng/ml IL-7, preferably 5 ng/ml and 0.1
to 1 .mu.g/ml PHA, preferably 0.5 .mu.g/ml, respectively. Such a
T-cell clone generated in vitro is suitable for in vivo transfer
into a mammal, preferably into a human, for the prevention,
treatment and/or diagnosis of cancer, preferably melanoma and other
MCSP expressing tumours such as breast cancer, notably lobular
breast carcinoma, astrocytoma, glioma, glioblastoma, neuroblastoma,
sarcoma and certain types of leukaemia, in particular melanoma.
[0094] A preferred aspect of embodiment (9) of the present
invention is a mature DC loaded with anyone of the peptides
according to embodiment (1) or fusion proteins of embodiment (2) of
the invention. Preferably the mature loaded DC is obtainable by the
method according to the invention described above.
[0095] A preferred aspect of embodiment (10) is a CD4.sup.+ T-cell
clone specific for anyone of the MCSP peptides of embodiment (1) or
fusion proteins of embodiment (2). Preferred is a CD4.sup.+ T-cell
clone obtainable by the method of embodiment (8) of the invention
described above.
[0096] An antibody specific for anyone of the peptides of
embodiment (1) described above is a preferred aspect of embodiment
(11) of the present invention. Such antibodies can be produced by
methods generally known in the art (see, e.g., Sambrook et al. in
"Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Press,
Plainview, N.Y. (1989)).
[0097] A preferred aspect of embodiment (12) is a composition
comprising anyone of the MCSP peptides or fusion proteins according
to embodiment (1) or (2) of the present invention, the nucleic
acids according to embodiment (3) of the invention, the vectors
according to embodiment (4) of the invention, the
transformed/transfected cells according to embodiment (5) of the
invention, the loaded mature DCs according to embodiment (9) of the
invention, CD4.sup.+ and CD8.sup.+ T-cell clones according to
embodiment (10) of the invention and/or antibodies according to
embodiment (11) of the invention, and a pharmaceutically acceptable
carrier. Said composition may be (and is preferably) a vaccine
further comprising an adjuvant.
[0098] It is also an embodiment of the present invention to use
anyone of the MCSP peptides or fusion proteins, the nucleic acid
molecules and/or the vector molecules, described above for the
preparation of immune cells, such as artificial APCs, mature DCs
loaded with anyone of the peptides according to the invention,
CD4.sup.+ and CD8.sup.+ T-cell clones specific for anyone of the
peptides according to the invention, B-cells secreting antibodies
specific for anyone of the MCSP peptides or fusion proteins
according to the invention and/or hybridomas secreting antibodies
specific for anyone of the MCSP peptides or fusion proteins
according to the invention. Said preparation of immune cells may be
performed ex vivo and in vivo. Ex vivo preparation is
preferred.
[0099] A further embodiment is the use of anyone of the MCSP
peptides according to the invention as a diagnostic marker for
cancer, preferably as a diagnostic marker for melanoma and other
MCSP expressing tumours such as breast cancer, notably lobular
breast carcinoma, astrocytoma, glioma, glioblastoma, neuroblastoma,
sarcoma and certain types of leukaemia, in particular for melanoma.
The MCSP peptides according to the invention can be found on the
cell surface of tumour cells, as well as on immune cells, if an
immune reaction against the tumour is already raised. Thus, the
MCSP peptides are suitable markers for in vivo and ex vivo imaging
and/or detection of tumours.
[0100] Also an embodiment of the invention is the use of anyone of
the MCSP peptides, the nucleic acid molecules and/or the vector
molecules according to the invention, for the manufacturing of a
medicament stimulating the production of protective antibodies
and/or immune cells. Said medicament is particular useful for
raising T-cell responses, especially CD4.sup.+ or CD8.sup.+ T cell
responses. A consequence of administration of said medicament may
be an immunization of the patient against the full length MCSP
protein of SEQ ID NO:1 or fragments thereof.
[0101] Also an embodiment is the use of anyone of the MCSP peptides
or fusion proteins, nucleic acid molecules, vector molecules,
loaded mature DCs, CD4.sup.+ T-cell clones and/or the antibodies
according to the invention, for the preparation of a medicament for
preventing, treating and/or diagnosing cancer, preferably for
preventing, treating and/or diagnosing melanoma, including
cutaneous and ocular melanoma, and other MCSP expressing tumours
such as breast cancer, notably lobular breast carcinoma,
astrocytoma, glioma, glioblastoma, neuroblastoma, sarcoma and
certain types of leukaemia, more preferably melanoma. Such a
medicament may function as a vaccine.
[0102] Furthermore, embodiment (16) of the invention relates to an
ex vivo method for diagnosing and/or monitoring a disorder
characterized by the expression of anyone of the MCSP peptides
according to the invention, comprising the following steps: (i)
contacting a biological sample, such as a biopsy, isolated from a
subject having or suspected to have said disorder, with an agent
that is specific for anyone of the peptides according to the
present invention, preferably the agent being a T-cell clone or an
antibody according to the invention; and (ii) determining the
interaction between the agent and the peptide. The term interaction
refers here to a binding interaction, which triggers the T-cell
clone to proliferate and to secrete cytokines, such as IFN-.gamma.
and/or TNF-.alpha..
[0103] A final embodiment is a method for treating a subject having
a disorder characterized by expression of anyone of the peptides
according to the present invention alone, or as part of the MCSP,
notably cancer. Said method comprises administering to the subject
an amount of anyone of the peptides, the nucleic acid, the vector,
the loaded mature DCs, the T-cell clone, the antibody, and/or
anyone of the compositions, all according to the present invention.
The amount to be administered will be determined by the medical
practitioner individually for each patient and depends on various
aspects including the type/severity of the disorder/cancer, the
mode of administration, the age and weight of the patient, etc.
[0104] In the method of embodiment (17), the cancer is preferably
selected from melanoma, including cutaneous and ocular melanoma,
and other MCSP expressing tumours such as breast cancer, notably
lobular breast carcinoma, astrocytoma, glioma, glioblastoma,
neuroblastoma, sarcoma and certain types of leukaemia.
[0105] In a first trial to arrive at the peptides according to the
invention, candidate peptides with 16 amino acids were chosen by
screening for binding motifs for HLA-DP4 within the core protein of
MCSP, and afterwards synthesized. HLA-DP-4 was selected because it
is the most frequent HLA-class-II-molecule in Caucasians
(expression by approx. 70%). Unfortunately, there are so far no
complete algorithms to predict HLA-DP-4 binding motifs within a
given protein sequence, as it already exists e.g. for
HLA-DR-molecules. So the design of peptides was based on anchor
motifs published before (amino acids F, L, Y or M for relative
position 1; F, L, Y, A for position 7 and V, Y, I for position 10)
or common sequences (e.g. MAGE-3.DP4-epitope). The following
peptides were selected and synthesized: SQVLFSVTRGAHYGEL (SEQ ID
NO:12), VRYLSTDPQHHAYDTV (SEQ ID NO:13), GEALVNFTQAEVYAGN (SEQ ID
NO:14) and PHEVSVHINAHRLEIS (SEQ ID NO:15).
[0106] In order to test the ability of the above peptides to be
T-cell stimulatory, DCs were derived from monocytes of healthy
donors using IL-4 und GM-CSF and loaded with 10 .mu.g/ml of
candidate peptide for 1 hour. Autologous CD4.sup.+ T-cells were
stimulated in 96-well-plates with those peptide-loaded DCs. After
restimulation on day 7, 14 and 21 the existence of peptide-specific
T-cells was tested by an ELISA technique. FIG. 1 shows
schematically the experimental protocol. Although various
experiments were performed, no peptide-specific CD4.sup.+ T-cells
were induced. Obviously, the peptides with SEQ ID NO:12 to 15 do
not represent naturally processed peptides recognized by T-cells.
Accordingly the approach was changed to yield optimal benefit from
the professional antigen processing and presentation by dendritic
cells.
[0107] The complete MCSP core protein sequence was screened by
computer algorithms (http://www.uni-tuebingen.de/uni/kxi, using the
database SYFPEITHI), based on experimental cleavage data for the
existence of peptides binding to different HLA-DR-molecules. A
region was identified in which several HLA-DR-binding candidate
antigens are localized, and a peptide with a length of 42 amino
acids was synthesized. DCs were loaded with this long peptide
overnight, so that potential T-cell epitopes within this peptide
could be processed and presented by the cell. DCs were matured 6 h
after peptide-loading by a cytokine cocktail (IL-1.beta., IL-6,
TNF-alpha, PGE.sub.2). CD4.sup.+ T-cells were stimulated with those
protein-loaded mature DCs on the next day. FIG. 2 shows
schematically the experimental protocol. Using this approach, 2
microcultures with peptide-specific CD4.sup.+ T-cells were
generated and cloned by limiting dilution. In total, 3
peptide-specific T-cell clones were obtained from microculture C2.
Further experiments were performed with clone C2/25, from hereon
called in short C25. This clone showed a specific IFN-.gamma.
production after stimulation with peptide-loaded autologous
EBV-B-cells, but not after stimulation with EBV-B-cells with or
without a control peptide (FIG. 3).
[0108] To determine the core epitope recognized by clone 25 within
the 42-mer, a set of 16-mer peptides, overlapping each other by 12
amino acids, was tested for recognition. Clone 25 recognized two
overlapping 16-mer peptides, namely ETNAVGQDVSVLFRVT (SEQ ID NO:8)
and VGQDVSVLFRVTGALQ (SEQ ID NO:9); (FIG. 4), but peptide
VGQDVSVLFRVTGALQ (SEQ ID NO:9) was found to stimulate clone 25 most
efficiently (FIG. 4). In a second step a set of truncated peptides
derived from the sequence of the two overlapping peptides was
tested to define the fine specificity of clone 25. The peptides
were truncated for up to 7 amino acids at the N- or C-terminus.
Truncations for up to 3 amino acids at both N- and C-terminus were
tolerated. Truncations going beyond either D at amino acid position
696 (SEQ ID No. 1) at the N or the A at amino acid position 706
resulted in loss of recognition by the T cell clone (FIG. 5), as
measured by an IFN-.gamma. ELISA. Further titration experiments
were carried out with the 16-mer MCSP-peptide VGQDVSVLFRVTGALQ (SEQ
ID NO:9) (amino acids 693-708 of SEQ ID No. 1) and a concentration
of 2 .mu.g/ml was found optimal to achieve CD4.sup.+ T-cell
stimulation. At 2 .mu.g/ml of the peptide, the CD4.sup.+ T-cells
secreted more than 4,000 .mu.g/ml IFN-.gamma.. This further
confirmed that VGQDVSVLFRVTGALQ (SEQ ID NO:9) is very efficiently
recognized by clone 25. Therefore this peptide was used in all
further experiments.
[0109] To analyze the HLA restriction of clone 25, it was tested
whether monoclonal anti-DR, anti-DQ or anti-DP antibodies would
inhibit the recognition of antigen-presenting cells by the clone.
The recognition of autologous stimulator EBV-B cells loaded with
MCSP peptide VGQDVSVLFRVTGALQ (SEQ ID NO:9) (amino acids 693-708 of
SEQ ID NO:1) was completely abolished by an anti-HLA-DR antibody,
while antibodies against HLA-DP or HLA-DQ had no influence (FIG.
6), as determined by the IFN-.gamma. production. Blood donor 4800,
from whom the stimulator cells were derived, was typed
HLA-DRB1*11/DRB1*03. In order to determine which allele clone 25 is
restricted to, peptide VGQDVSVLFRVTGALQ (SEQ ID NO:9) was loaded on
several EBV-B cell lines with different known HLA class II typings.
In particular EBV-B cell lines LP2, LB1981-, R12-, PV6-, AC 42- and
4800-EBV were used, of which only PV6-, AC 42- and 4800-EBV are
HLA-DR11 positive. It was found that only those expressing
HLA-DRB1*11 (PV6-, AC 42- and 4800-EBV) were able to present the
peptide to clone 25, as determined by IFN-.gamma. production (FIG.
7). To establish that clone 25 reacted specifically with the MCSP
peptide VGQDVSVLFRVTGALQ (SEQ ID NO:9) and its reaction was not
caused by a contaminant within the peptide preparation or a
chemical modification of the synthesized peptide, EBV-B cells
stimulator cells were retrovirally transfected with the fusion
peptide Ii comprising the amino acid residues 392-748 of MCSP,
giving rise to the processed peptide VGQDVSVLFRVTGALQ (SEQ ID
NO:9). For that, HLA-DR11 positive (4800- and MVGS EBV) and
HLA-DR11 negative cells (MMDH EBV) were transduced with a
retroviral construct retro-Ii.MCSP, which encodes a truncated human
invariant chain (Ii) fused with a truncated MCSP (amino acid
residues 392-748). The fusion guarantees an endosomal targeting and
therefore effective processing and HLA class II presentation. Clone
25 reacted only with the recombinantly loaded HLA-DR11 positive
cells, demonstrating that the clone was really MCSP-specific and
not directed against a contaminant in the batch of the peptide
VGQDVSVLFRVTGALQ (SEQ ID NO:9) (FIG. 8). From previous studies it
was known, that tumour-specific CD4.sup.+ T cells can directly
recognize HLA class II-expressing tumour cells. Therefore the
ability of clone 25 to recognize directly MCSP-expressing melanoma
cell lines was tested, as shown in FIG. 9. MCSP-positive melanoma
cells expressing HLA-DRB1*11, namely ER-MEL-3 and ER-MEL-4,
stimulated clone 25 to produce IFN-.gamma., whereas HLA-DR 11
negative cells, namely MEL 397, LB 1622, did not stimulate clone
25.
[0110] In conclusion, the aim set out at the beginning, namely to
identify and characterize an MCSP-T-cell epitope was achieved. That
way the hypothesis about the existence of MCSP-specific T-cell
responses was impressively affirmed. MCSP-specific CD4.sup.+
T-cells were isolated and it was shown that endogenously processed
antigen is recognized by said cells, which respond to the antigen
by production of high amounts of IFN-.gamma.. Moreover, it was
demonstrated that the MCSP-specific CD4.sup.+ T-cells recognize
tumour cells expressing MCSP. It is postulated therefore, that said
cells play an important role in anti-tumour immunity in vivo. The
identified peptide according to the invention can be used directly
in immunotherapy against cancer, especially against malignant
melanoma, in form of a medicament comprising said peptides. Said
medicament may be used for peptide vaccination and/or for
administering DCs loaded with anyone of the peptides according to
the invention. It was shown here that MCSP and the peptide
fragments derived therefrom are promising candidate antigens for
immunotherapy, because they are expressed in >90% of melanoma.
Considering the functional role of MCSP in growth, invasion and
metastasis of malignant melanoma, a vaccination against MCSP could
be useful, especially in an adjuvant setting after excision of
larger primary tumours or regional lymph node metastasis. Phase I
clinical studies involving vaccinating melanoma patients with the
peptide-loaded DCs according to the invention are already
planned.
[0111] In the passages above the identification of a MCSP CD4.sup.+
T helper cell epitope using dendritic cells loaded with a long
candidate peptide as stimulator cells for CD4.sup.+ T cells was
described. In further experiments another long peptide from the
MCSP core protein sequence where computer algorithms had predicted
several HLA-DR binding motifs and in addition a HLA-A2 binding
motif was identified. This long peptide represented amino acids
1270-1300 (PPADIVFSVKSPPSAGYLVMVSRGALADEPP; SEQ ID NO:36) of MCSP.
In order to identify both CD4.sup.+ and CD8.sup.+ T cell epitopes,
said peptide was modified so that the peptide could enter the
cytosol of the dendritic cells to get access to the HLA class I
presentation pathway as follows: It has been shown that the protein
transduction domain (PDT) embedded in the HIV TAT protein (amino
acids 47-57=YGRKKRRQRRR; SEQ ID NO:52) can successfully mediate the
introduction of peptides and proteins into mammalian cells in vitro
and in vivo. Furthermore, it has been demonstrated that
structurally modified nonnaturally occurring peptide variants can
be even more efficient in protein transduction than the original
wild type TAT PDT (Ho, A., et al., Cancer Research, 61: 474-477
(2001)). Therefore a TAT PDT variant peptide (YARAAARQARA; SEQ ID
NO:53) was placed at the N-terminus of the above MCSP peptide
resulting in a 42-mer with the following sequence:
YARAAARQARAPPADIVFSVKSPPSAGYLVMVSRGALADEPP (SEQ ID NO:54) ("long
TAT-MCSP-peptide").
[0112] Dendritic cells (DC) were derived from monocytes of healthy
donors using IL-4 and GM-CSF and loaded with the long peptide of
SEQ ID NO:54 at 20 .mu.g/ml overnight so that potential T-cell
epitopes within this peptide could be processed and presented by
the cell. DCs were matured 6 h after peptide-loading by a cytokine
cocktail (IL-1.beta., IL-6, TNF-.alpha., PGE.sub.2) as described in
Example 3 (b). Autologous CD4.sup.+ T-cells were stimulated with
protein-loaded mature DCs in 96-well-plates on the next day (FIG.
10). After restimulation on day 7, 14 and 21 (compare Example 4)
the existence of peptide-specific T-cells was tested by ELISA
technique (compare Example 5). FIG. 10 shows schematically the
experimental protocol.
[0113] 3 microcultures with peptide-specific CD4.sup.+ T-cells
could be generated and were cloned by limiting dilution (Example
5). 3 peptide-specific T-cell clones could be generated from
microculture F1 in total. Further experiments were performed with
clone F1/3. This clone showed a specific Interferon-.gamma.
production after stimulation with peptide-loaded autologous
EBV-B-cells, but not after stimulation with EBV-B-cells with or
without a control peptide (FIG. 11).
[0114] To determine the core epitope recognized by clone 3 within
the 42-mer of SEQ ID NO:54, a set of overlapping peptides including
the TAT PDT variant peptide was tested for recognition (Example 6).
Clone 3 recognized two overlapping 16-mer peptides within the MCSP
sequence but not the modified TAT peptide (FIG. 12). Peptide
PPSAGYLVMVSRGALA (SEQ ID NO:42) was found to stimulate clone 3 most
efficiently.
[0115] In a second step a set of truncated peptides derived from
the sequence of peptide PPSAGYLVMVSRGALA (MCSP.sub.1281-1296; SEQ
ID NO:42) was tested to define the fine specificity of clone 3
(Example 7). Truncation of either G at the N terminus or L at the
C-terminus resulted in loss of recognition by the T cell clone
(FIG. 13). The 12-mer peptide GYLVMVSRGALA (MCSP.sub.1285-1296; SEQ
ID NO:46) turned out to be the shortest peptide efficiently
recognized.
[0116] To analyze the HLA restriction of clone 3, it was tested
whether monoclonal anti-DR, anti-DQ or anti-DP antibodies would
inhibit the recognition of antigen-presenting cells by the clone
(Example 8). The recognition by clone 3 of autologous EBV-B cells
loaded with peptide MCSP.sub.1281-1296 (SEQ ID NO:48) was abolished
by an anti-HLA-DR antibody, while antibodies against HLA-DP or
HLA-DQ had no influence (FIG. 14).
[0117] Blood donor 11325 was typed HLA-DR 11/13. To determine to
which allele clone 3 is restricted to, peptide PPSAGYLVMVSRGALA
(SEQ ID NO:42) was loaded on several EBV-B cell lines with
different known HLA class II typings, and only those expressing
HLA-DR 11 were able to present the peptide to clone 3 (FIG. 15;
Example 9).
[0118] Since it has been shown that tumor-specific CD4.sup.+ T
cells can directly recognize HLA class II-expressing tumor cells,
the direct recognition of MCSP-expressing melanoma cell lines by
clone 3 was assayed (Example 11). MCSP-positive melanoma cells
expressing HLA-DR 11 (ER-MEL-4) stimulated clone 3 to produce
IFN-.gamma., whereas HLA-DR 11 negative cells did not, as shown in
FIG. 16.
[0119] In summary, using an approach with a TAT PDT variant peptide
fused to the MCSP candidate peptide MCSP-specific CD4.sup.+ T cell
clones were generated which recognize a different MCSP peptide
presented by HLA-DR11 on human melanoma cells.
[0120] In order to isolate MCSP specific CD8.sup.+ T cells, DCs
were derived from monocytes of healthy donors using IL-4 and GM-CSF
and loaded with the long TAT-MCSP-peptide of SEQ ID NO:54 at 20
.mu.g/ml overnight so that potential T-cell epitopes within this
peptide could be processed and presented by the cell. DCs were
matured 6 h after peptide-loading by a cytokine cocktail
(IL-1.beta., IL-6, TNF-.alpha., PGE.sub.2) as described in Example
3 (b). Autologous CD8.sup.+ T-cells were stimulated with
protein-loaded mature DCs in 96-well-plates on the next day (FIG.
17). After restimulation on day 7, 14 and 21 (compare Example 4)
the existence of peptide-specific T-cells was tested by ELISA
technique (compare Example 5). FIG. 17 shows schematically the
experimental protocol.
[0121] Furthermore, CD8.sup.+ T-cells from microcultures showed a
specific IFN-.gamma. production after stimulation with autologous
EBV-B-cells of donor 11325 loaded with the long TAT-MCSP-peptide
(SEQ ID NO:54; 10 .mu.M), but not after stimulation with EBV-V
cells with a control peptide (FIG. 18; Example 5).
[0122] The invention is further described by the following examples
which are, however, not to be construed as to limit the
invention.
EXAMPLES
[0123] 1. Computer Algorithms: The complete MCSP core protein
sequence was screened by computer algorithms
(http://www.uni-tuebingen.de/uni/kxi, using the database
SYFPEITHI). Various HLA I/II alleles were chosen, inter alia DR
0101, DR 0301, DR 0401, DR 0701, DR 1101, DR 1501. Candidate
peptides were predicted for the various DR molecules.
[0124] 2. Peptid synthesis: Peptides were synthesized using F-moc
for transient NH.sub.2-terminal protection and were characterized
using mass spectrometry. All peptides were >80% pure as
indicated by analytical HPLC. Lyophilized synthetic peptides were
dissolved in DMSO (Merck)/acetic acid (10 mM) and stored at
-20.degree. C. Peptides were purchased from Coring System
Diagnostix GmbH (Gernsheim, Germany).
3. Preparation of Peptide Loaded Mature DCs and CD4.sup.+
T-Cells:
[0125] (a) Isolation of immature DCs and CD4.sup.+ T-responder
cells: Peripheral blood mononuclear cells (PBMCs) were isolated
from leukapheresis products obtained from healthy donors after
informed consent. First, PBMCs were isolated by centrifugation on
Lymphoprep (Nycomed Pharma, Oslo, Norway). In order to minimize
contamination of PBMCs with platelets, the preparation was first
centrifuged for 20 min/1,000 rpm at room temperature. After removal
of the top 20-25 ml, containing most of the platelets, the tubes
were centrifuged for 20 min/1,500 rpm at room temperature. The
interphase containing the PBMCs was harvested and then 3.times.
washed (or more) in cold phosphate buffer solution with 2 mM EDTA
in order to eliminate any remaining platelets.
[0126] PBMCs were plated in 85 mm tissue culture dishes (Falcon.
Cat. No. 3003; Becton Dickinson, Hershey, USA) at a density of
50.times.10.sup.6 cells per dish in 10 ml RPMI 1640, supplemented
with 1% heat-inactivated autologous plasma, 2 mM L-glutamine and 20
.mu.g/ml gentamicin, hereafter referred to as complete DC medium,
and incubated at 37.degree. C. and 5% CO.sub.2 for 2 h. The
non-adherent fraction was removed and frozen and 10 ml of complete
DC medium was added to the adherent cells. On day 1 and 3 1,000
U/ml GM-CSF and 800 U/ml IL-4 (both from CellGenix) were added to
the cultures to induce the differentiation of the adherent
monocytes. On day 5 the non-adherent cells were used as a source of
enriched immature DCs. Immature DCs were then loaded with the MCSP
42-mer peptide (either SEQ ID NO:3 or SEQ ID NO:54) at 50 .mu.g/ml
(SEQ ID NO:3) or 20 .mu.g/ml (SEQ ID NO:54) overnight in complete
DC medium and after 6 h maturation was induced by adding IL-1.beta.
(10 ng/ml), IL-6 (100 U/ml), TNF-.alpha. (10 ng/ml) and PGE.sub.2
(1 .mu.g/ml) to the culture medium (see also below under (b)). The
next day the non-adherent fraction was thawed and CD4.sup.+ T
lymphocytes were isolated by positive selection using an anti-CD4
monoclonal antibody coupled to microbeads (Miltenyi Biotech,
Bergisch Gladbach, Germany) (for more details see section
above).
[0127] (b) Peptide loading/pulsing of immature DCs and maturation:
Immature DCs (1.times.10.sup.5) were incubated at 37.degree. C., 5%
CO.sub.2, for 1 h or 20 h (overnight) in RPMI medium supplemented
with 1% human serum, IL-4 (100 U/ml) and GM-CSF (100 ng/ml) and
TNF-.alpha. (1 ng/ml) in the presence of MCSP-derived peptides or
control peptides at concentrations of between 2-50 .mu.g/ml. In
case the DCs were incubated overnight, the DCs received a
maturation stimulus after 6 h by addition of IL-1.beta. (10 ng/ml),
IL-6 (100 U/ml), TNF-.alpha. (10 ng/ml) and PGE.sub.2 (1 .mu.g/ml)
to the culture medium. The pulsed/loaded DCs were washed and
subsequently used in the stimulation assay.
4. T-Cell Induction Assay:
[0128] (A) CD4.sup.+ T-cell induction: On the day of stimulation
(day 0) loaded DCs were washed and added at 1.times.10.sup.4 per
round-bottom microtiter dish well to 10.sup.5 CD4.sup.+ T-cells in
200 .mu.l complete T-cell medium in the presence of IL-6 (1000
U/ml), IL-12 (10 ng/ml) and TNF-.alpha. (1 ng/ml). The CD4.sup.+
T-cells were weekly (days 7, 14 and 21) restimulated with
autologous DCs freshly loaded/pulsed with the MCSP peptide and were
grown in complete T-cell medium supplemented with IL-2 (10 U/ml),
IL-7 (5 ng/ml).
[0129] (B) CD8.sup.+ T cell induction: On the day of stimulation
(day 0) loaded DCs were washed and added at 1.5.times.10.sup.4 per
round-bottom microtiter dish well to 1.5.times.10.sup.5 CD8.sup.+
T-cells of donor 11325 in 200 .mu.l complete T-cell medium in the
presence of IL-6 (1000 U/ml), IL-12 (10 ng/ml) and TNF-.alpha. (1
ng/ml). The CD8.sup.+ T-cells were weekly (days 7, 14 and 21)
restimulated with autologous DCs freshly loaded/pulsed with the
MCSP peptide and were grown in complete T-cell medium supplemented
with IL-2 (10 U/ml), IL-7 (5 ng/ml).
[0130] 5. IFN-.gamma. production by stimulated CD4.sup.+ and
CD8.sup.+ T cells; Obtention of CD4.sup.+ T-cell lines and clones
specific for the MCSP peptide: The microcultures of stimulated
CD4.sup.+ or CD8.sup.+ T-cells (see Example 4) were assessed on day
30 after start of the culture for their capacity to produce
IFN-.gamma. when stimulated with autologous EBV-B cells
pulsed/loaded with the MCSP peptide. Autologous EBV-B cells
(5.times.10.sup.5) were incubated for 18-20 h at 37.degree. C. in
the presence of 10 .mu.g/ml or 20 .mu.g/ml (compare Figure legends)
of the MCSP peptide or an irrelevant control peptide as a negative
control. EBV-B-cells referred to herein are B-cells which were
immortalized with Epstein Barr virus. The EBV-B-cells were prepared
according to art-standard procedures. Peptide-pulsed EBV-B cells
were washed and added at 1.5.times.10.sup.4 cells per round-bottom
well to 4.times.10.sup.3 CD4.sup.+ or CD8.sup.+ T-cells in 100
.mu.l of complete T-cell medium supplemented with IL-2 (25 U/ml).
After 18-20 h, supernatants were harvested and assessed for
IFN-.gamma. content using an ELISA assay with reagents from
Medgenix Diagnostics-Biosource (Fleurus, Belgium). Briefly the
assay is a standard ELISA in which IFN-.gamma. antibodies were
coated onto the wells of plastic microtiter plates prior to
incubation with cell supernatants to determine the amount of
IFN-.gamma. produced, with a specificity of 20-4,000 pg/ml.
Cytokine secretion was considered significant if it was at least
two-fold above the background response of T-cells to EBV-B cells
pulsed/loaded with the control peptide, and if it exceeded 500 pg
of IFN-.gamma. per ml. Often the IFN-.gamma. production was found
to overshoot the upper specificity range of the ELISA, and then the
amount of IFN-.gamma. was said to be >4,000 .mu.g/ml.
[0131] The CD4.sup.+ T-cell lines producing IFN-.gamma., i.e. those
which recognize the MCSP peptide, were cloned by limiting dilution.
Three CD4.sup.+ T-cell clones were obtained and grown in complete
T-cell medium supplemented with IL-2 (50 U/ml), IL-7 (5 ng/ml). The
clones were supplemented with fresh culture medium once a week and
passaged with EBV-B stimulator cells pulsed/loaded with the MCSP
peptide at 1-2 week intervals. Clone C2/25 was selected for all
further experiments on the peptide with SEQ ID NO:3. Further
experiments on the peptide with SEQ ID NO:54 were performed with
clone F1/3.
[0132] 6. Identification of MCSP HLA-DR restricted peptide: In
order to identify the core epitope recognized by CD4.sup.+ T-cell
clone C2/25 ("25"), 14 to 16 amino acid peptides (SEQ ID NOs:3-10)
corresponding to overlapping parts of the MCSP peptide with SEQ ID
NO:3 were synthesized and loaded onto autologous EBV-B cells and
tested for recognition (FIG. 4).
[0133] To determine the core epitope recognized by clone F1/3 ("3")
within the 42-mer of SEQ ID NO:54, a set of overlapping peptides
including the TAT PDT variant peptide was tested for recognition
(FIG. 12).
[0134] Peptides were synthesized using F-moc for transient
NH.sub.2-terminal protection and were characterized using mass
spectrometry. All peptides were >80% pure as indicated by
analytical HPLC. Lyophilized synthetic peptides were dissolved in
DMSO (Merck)/acetic acid and used at a final concentration of 1
.mu.M/ml. EBV-B cells (1.5.times.10.sup.4 per round-bottomed
microtiter plate well) were incubated at 1 h at 37.degree. C., 8%
CO.sub.2 in the presence of the different peptides. The CD4.sup.+
T-cell clone 25 or 3, respectively, was then added at
4.times.10.sup.3 per well. Assay medium was complete T-cell medium
supplemented IL-2 (25 U/ml). After 18-20 h, supernatants were
harvested and assessed for IFN-.gamma. production using an ELISA
assay.
[0135] The two overlapping peptides ETNAVGQDVSVLFRVT (SEQ ID NO:8)
and VGQDVSVLFRVTGALQ (SEQ ID NO:9) were found to stimulate clone
25, whereby the peptide with SEQ ID NO:9 had the most stimulatory
effect. Clone 3 recognized two overlapping 16-mer peptides within
the MCSP sequence but not the modified TAT peptide (FIG. 12).
Peptide PPSAGYLVMVSRGALA (SEQ ID NO:42) was found to stimulate
clone 3 most efficiently.
[0136] 7. Determination of minimal peptides still able to stimulate
CD4.sup.+ T-cell clone 25 or 3: Unlike HLA class I restricted
peptides, class II peptides vary considerably in length and can
tolerate truncation and extension at both N- and C-termini.
Therefore the ability of truncated peptides (LFRVTGALQ (SEQ ID
NO:22), VLFRVTGALQ (SEQ ID NO:23), SVLFRVTGALQ (SEQ ID NO:24),
VSVLFRVTGALQ (SEQ ID NO:25), DVSVLFRVTGALQ (SEQ ID NO:26),
QDVSVLFRVTGALQ (SEQ ID NO:27), GQDVSVLFRVTGALQ (SEQ ID NO:28),
VGQDVSVLFRVTGAL (SEQ ID NO:29), VGQDVSVLFRVTGA (SEQ ID NO:30),
VGQDVSVLFRVTG (SEQ ID NO:31), VGQDVSVLFRVT (SEQ ID NO:32),
VGQDVSVLFRV (SEQ ID NO:33), VGQDVSVLFR (SEQ ID NO:34), VGQDVSVLF
(SEQ ID NO:35) to stimulate the CD4.sup.+ T-cell clone 25 was
compared to the untruncated peptide VGQDVSVLFRVTGALQ (SEQ ID NO:9).
In essence EBV-B cells (5.times.10.sup.3 per round-bottomed
microtiter plate well) were incubated at 2 h at 37.degree. C., 8%
CO.sub.2 in the presence of the different peptides. The CD4.sup.+
T-cell clone 25 was then added at 2.5.times.10.sup.3 per well.
Assay medium was complete T-cell medium supplemented with IL-2 (25
U/ml). After 18-20 h, supernatants were harvested and assessed for
IFN-.gamma. production using an ELISA assay. The results are
summarized in FIG. 5.
[0137] Analogously, a set of truncated peptides derived from the
sequence of peptide PPSAGYLVMVSRGALA (MCSP.sub.1281-1296; SEQ ID
NO:42) was tested to define the fine specificity of clone 3.
Truncation of either G at the N terminus or L at the C-terminus
resulted in loss of recognition by the T cell clone (FIG. 13). The
12-mer peptide GYLVMVSRGALA (MCSP.sub.1285-1296; SEQ ID NO:46)
turned out to be the shortest peptide efficiently recognized.
[0138] 8. Determination of HLA restriction element utilized by
MCSP-specific CD4.sup.+ T-cell clone 25 and 3: To analyze the HLA
restriction of CD4.sup.+ T-cell clone 25 and 3, three monoclonal
antibodies directed against anti-DR (L243; BD Biosciences, San
Jose, USA), anti-DQ (CSPVL3; Immunotech) or anti-DP (B7/21; BD
Biosciences, San Jose, USA) were used to test which one inhibited
the recognition of antigen-presenting cells by the clone. First
EBV-B cells (1.5.times.10.sup.4 per round-bottomed microtiter plate
well) were incubated for 1 h at 37.degree. C., 8% CO.sub.2 in the
presence of the peptide (SEQ ID NO:9 for clone 25, SEQ ID NO:48 for
clone 3). The antibodies were added at a concentration of 5 ng/ml.
The CD4.sup.+ T-cell clone 25 or 3 was then added at
4.times.10.sup.3 per well. Assay medium was complete T-cell medium
supplemented with IL-2 (25 U/ml). After 18-20 h, supernatants were
harvested and assessed for IFN-.gamma. production using an ELISA
assay.
[0139] 9. Determination of HLA-DR restriction element utilized by
MCSP-specific CD4.sup.+ T-cell clone 25: It was determined that
cytokine secretion by the CD4.sup.+ T-cell clone 25 occurred in
response to EBV-B cells pulsed/loaded with MCSP-derived peptides
restricted to HLA-DR. To further define the HLA-restriction element
utilized by clone 25, additional EBV-B cell lines were used for
peptide presentation as described above. In particular LP2-,
LB1981-, R12-, PV6-, AC 42- and 4800-EBV which have with different
HLA class II molecules (HLA-DR11 positive or HLA-DR11 negative)
were used. They were pulsed/loaded for 1 h with 1 .mu.M MCSP
peptide VGQDVSVLFRVTGALQ (SEQ ID NO:9) and tested for recognition
by the CD4.sup.+ T cell clone. As control, the cells were pulsed in
the absence of protein. IFN-.gamma. production of the CD4.sup.+ T
cells was measured after overnight co-culture (20 h) by ELISA. The
results are summarized in FIG. 7.
[0140] 10. Preparation and use of MCSP peptide fusion protein: An
EBV-B cell line was transduced with the retroviral construct
retro-Ii.MCSP, which encodes a truncated human invariant chain (Ii)
fused with a truncated MCSP (amino acid residues 392-748). The
construct was kindly provided by Professor Gerd Pluschke, Swiss
Tropical Institute, Basel, Switzerland.
(a) Plasmids and cloning of fusion constructs: The MCSP cDNA and
its corresponding polypeptide are set forth in SEQ ID Nos:1 and 2,
respectively. The plasmid comprising the human invariant chain (Ii)
cDNA, named lipSV51L, was kindly provided by Dr. J. Pieters (Basel
Institute for Immunology, Basel, Switzerland; J. Cell Science 106,
831-846 (1993)). The plasmid pMFG was kindly provided by Dr. O.
Danos (Somatrix Therapy Corporation, Alameda, Calif. USA) The MCSP
cDNA coding for amino acid residues 659-735 (SEQ ID NO:1) was
transferred into the pMFG vector after introduction of appropriate
restriction enzyme recognition sites at the 5' and 3' end of the
coding sequence. A BamHI restriction site was introduced at the 5'
end and at the 3' end, by PCR using the forward primers
5'-GGGGATCCCATCCGGCCGGCCATACAG-3' (SEQ ID NO:16) and the reverse
primer: 5'-GGGGATCCTCACCGCTGGTGGAACGCCTGTG-3' (SEQ ID NO:17); the
BamHI restriction is shown in italic, and the stop codon is
underlined. The PCR product was cloned into a pCR2.1 vector and
sequenced according to standard methods. The BamHI-BamHI
amplification product was cloned into pMFG opened with the enzyme
BamHI, to give rise to pMFG-MCSP.
[0141] The cDNA encoding the amino terminal end (i.e. the
cytoplasmic tail and the transmembrane region) of the human
invariant chain polypeptide (hu-Ii; residues 1-80) was amplified by
PCR using IipSV51L as template. The following primers were used:
hu-Ii sense: 5'-TTTCCATGGATGACCAGCGCGAC-3' (SEQ ID NO:18); and
hu-Ii antisense: 5'-TTTGGATCCGGAAGCTTCATGCGCAGGTTC-3' (SEQ ID
NO:19); The recognition sites for NcoI and Bam HI are in italic.
The PCR product was cloned into pCR2.1 and sequenced according to
standard methods. The NcoI and BamHI amplification product was
cloned into pMFG, opened with the enzymes NcoI and BamHI resulting
in pMFG-Ii.
[0142] The recombinant plasmid pMFG-Ii was reopened with BamHI, and
the BamHI MCSP fragment isolated from pMFG-MCSP ligated. The
resulting plasmid gives rise to the fusion protein huIi.MCSP (in
short Ii-MCSP or Ii). Recombinant plasmids containing the MCSP
fragment in the right orientation were identified by restriction
fragment analysis.
[0143] The DNA sequence of huIi-MCSP (SEQ ID NO:20) (start and stop
codons are in bold; the huIi fragment is in small letters; the MCSP
fragment is in upper case; a detected sequence variation is
underlined; there the sequence differs from the published sequence
(TAGTA becomes GGGGG; since the sequence variation lies outside the
target area, experiments were continued with said construct):
TABLE-US-00004 atggaccttatctccaacaatgagcaactgcccatgctgggccggcgccc
tggggccccggagagcaagtgcagccgcggagccctgtacacaggctttt
ccatcctggtgactctgctcctcgctggccaggccaccaccgcctacttc
ctgtaccagcagcagggccggctggacaaactgacagtcacctcccagaa
cctgcagctggagaacctgcgcatgaagcttcccaaggatcccATCCGGC
CGGCCATACAGATCCACCGCAGCACAGGGTTGCGACTGGCCCAAGGCTCT
GCCATGCCCATCTTGCCCGCCAACCTGTCGGTGGAGACCAATGCCGTGGG
GCAGGATGTGAGCGTGCTGTTCCGCGTCACTGGGGCCCTGCAGTTTGGGG
AGCTGCAGAAGCAGGGGGCAGGTGGGGTGGAGGGTGCTGAGTGGTGGGCC
ACACAGGCGTTCCACCAGCGGTGA.
[0144] The corresponding amino acid sequence is as follows (SEQ ID
NO:21) (the huIi fragment is in small letters; the MCSP fragment is
in upper case; the detected sequence variation is underlined; there
the sequence differs from the published sequence (HST becomes QGA;
since the sequence variation lies outside the target area,
experiments were continued with said construct):
TABLE-US-00005 mdlisnneqlpmlgrrpgapeskcsrgalytgfsilvtlllagqattayf
lyqqqgrldkltvtsqnlqlenlrmklpkdpIRPAIQIHRSTGLRLAQGS
AMPILPANLSVETNAVGQDVSVLFRVTGALQFGELQKQGAGGVEGAEWWA TQAFHQRZ.
(b) Retroviral transduction of EBV cell lines: EBV-transformed B
cells with different HLA class II molecules (4800 EBV and MVGS EBV
as HLA-DR11 positive cells and MMDH EBV as HLA-Dr11 negative cells)
were infected by resuspending the cells in an infection cocktail
and centrifugation. Target cells were resuspended in 60 mm tissue
culture plates (Falcon) at a density of 1.times.10.sup.6 cells in 4
ml infection cocktail. The plates were centrifuged for 2 h at
32.degree. C. and 1,200 rpm in an ICE centrifuge, rotor type 228.
for each plate to be transduced, 4 ml of infection cocktail was
prepared by diluting the viral supernatant 1:2 in EBV B-cell growth
medium and adding protamine sulfate to a final concentration of 6
.mu.g/ml. Centrifugation was followed by another 2 h of incubation
in a humidified incubator at 37.degree. C. and cells were
transferred to 4 ml of target cell growth medium. This transduction
cycle was carried out immediately after plating the cells and was
repeated at 24 h and 48 h. The infected EBV-B cells were assayed
for EGFP reporter gene expression by FACS analysis 24 h to 48 h
following the third infection cycle.
[0145] (c) IFN-.gamma. production assay: 4.times.10.sup.3 CD4.sup.+
T-cells of clone 25 were washed and cultured overnight in the
presence of 5.times.10.sup.3 retrovirally transduced EBV B-cells
(4800 EBV, MVGS EBV and MMDH EBV), in 100 .mu.l complete T-cell
medium containing 25 U/ml recombinant human IL-2 in a round-bottom
96 well plate. All co-cultures were performed in triplicate. 50
.mu.l culture supernatant was assayed for the presence of
IFN-.gamma. by ELISA (IFN-.gamma. ELISA Biosource). Briefly, ELISA
plates precoated with anti-human IFN-.gamma. antibodies were washed
and incubated with 50 .mu.l of culture supernatant and 50 .mu.l of
biotinylated anti-human IFN-.gamma. antibody (1:1,250) for 2 h at
room temperature. Following three washes the plates were incubated
with 50 .mu.l per well horseradish peroxidase conjugated
streptavidin (1:3,000 in PBS with 0.5% BSA) for 30 min at room
temperature, which was detected by TMB substrate, and
H.sub.2SO.sub.4 to stop the reaction. The optical density was read
at 450 nm. Samples containing 4,000 .mu.g/ml IFN-.gamma. and 1:2
dilutions were used as standards.
[0146] 11. MCSP positive melanoma cells as stimulators for
CD4.sup.+ T-cell clone 25 and 3: In order to test whether the
CD4.sup.+ T-cell clones 25 and 3 can directly recognize HLA-DR11
positive melanoma cells, melanoma cells which were known to be
either HLA-DR11 positive or negative (MEL 397 (HLA-DR11 negative),
LB 1622 (HLA-DR11 negative), ER-MEL-3 (HLA-DR11 positive) and
ER-MEL-4 (HLA-DR11 positive) were preincubated at 2.times.10.sup.4
cells in flat bottom microwells for 24-48 h to reach a monolayer of
tumour cells. Clone 25 or 3, respectively, (4.times.10.sup.3
cells/well) was then added and after co-culturing for 20 h the
supernatant was tested for the presence of IFN-.gamma. by ELISA.
The results are shown in FIGS. 9 and 16.
Sequence CWU 1
1
5412322PRTHomo sapiens 1Met Gln Ser Gly Arg Gly Pro Pro Leu Pro Ala
Pro Gly Leu Ala Leu 1 5 10 15Ala Leu Thr Leu Thr Met Leu Ala Arg
Leu Ala Ser Ala Ala Ser Phe20 25 30Phe Gly Glu Asn His Leu Glu Val
Pro Val Ala Thr Ala Leu Thr Asp35 40 45Ile Asp Leu Gln Leu Gln Phe
Ser Thr Ser Gln Pro Glu Ala Leu Leu50 55 60Leu Leu Ala Ala Gly Pro
Ala Asp His Leu Leu Leu Gln Leu Tyr Ser65 70 75 80Gly Arg Leu Gln
Val Arg Leu Val Leu Gly Gln Glu Glu Leu Arg Leu85 90 95Gln Thr Pro
Ala Glu Thr Leu Leu Ser Asp Ser Ile Pro His Thr Val100 105 110Val
Leu Thr Val Val Glu Gly Trp Ala Thr Leu Ser Val Asp Gly Phe115 120
125Leu Asn Ala Ser Ser Ala Val Pro Gly Ala Pro Leu Glu Val Pro
Tyr130 135 140Gly Leu Phe Val Gly Gly Thr Gly Thr Leu Gly Leu Pro
Tyr Leu Arg145 150 155 160Gly Thr Ser Arg Pro Leu Arg Gly Cys Leu
His Ala Ala Thr Leu Asn165 170 175Gly Arg Ser Leu Leu Arg Pro Leu
Thr Pro Asp Val His Glu Gly Cys180 185 190Ala Glu Glu Phe Ser Ala
Ser Asp Asp Val Ala Leu Gly Phe Ser Gly195 200 205Pro His Ser Leu
Ala Ala Phe Pro Ala Trp Gly Thr Gln Asp Glu Gly210 215 220Thr Leu
Glu Phe Thr Leu Thr Thr Gln Ser Arg Gln Ala Pro Leu Ala225 230 235
240Phe Gln Ala Gly Gly Arg Arg Gly Asp Phe Ile Tyr Val Asp Ile
Phe245 250 255Glu Gly His Leu Arg Ala Val Val Glu Lys Gly Gln Gly
Thr Val Leu260 265 270Leu His Asn Ser Val Pro Val Ala Asp Gly Gln
Pro His Glu Val Ser275 280 285Val His Ile Asn Ala His Arg Leu Glu
Ile Ser Val Asp Gln Tyr Pro290 295 300Thr His Thr Ser Asn Arg Gly
Val Leu Ser Tyr Leu Glu Pro Arg Gly305 310 315 320Ser Leu Leu Leu
Gly Gly Leu Asp Ala Glu Ala Ser Arg His Leu Gln325 330 335Glu His
Arg Leu Gly Leu Thr Pro Glu Ala Thr Asn Ala Ser Leu Leu340 345
350Gly Cys Met Glu Asp Leu Ser Val Asn Gly Gln Arg Arg Gly Leu
Arg355 360 365Glu Ala Leu Leu Thr Arg Asn Met Ala Ala Gly Cys Arg
Leu Glu Glu370 375 380Glu Glu Tyr Glu Asp Asp Ala Tyr Gly His Tyr
Glu Ala Phe Ser Thr385 390 395 400Leu Ala Pro Glu Ala Trp Pro Ala
Met Glu Leu Pro Glu Pro Cys Val405 410 415Pro Glu Pro Gly Leu Pro
Pro Val Phe Ala Asn Phe Thr Gln Leu Leu420 425 430Thr Ile Ser Pro
Leu Val Val Ala Glu Gly Gly Thr Ala Trp Leu Glu435 440 445Trp Arg
His Val Gln Pro Thr Leu Asp Leu Met Glu Ala Glu Leu Arg450 455
460Lys Ser Gln Val Leu Phe Ser Val Thr Arg Gly Ala Arg His Gly
Glu465 470 475 480Leu Glu Leu Asp Ile Pro Gly Ala Gln Ala Arg Lys
Met Phe Thr Leu485 490 495Leu Asp Val Val Asn Arg Lys Ala Arg Phe
Ile His Asp Gly Ser Glu500 505 510Asp Thr Ser Asp Gln Leu Val Leu
Glu Val Ser Val Thr Ala Arg Val515 520 525Pro Met Pro Ser Cys Leu
Arg Arg Gly Gln Thr Tyr Leu Leu Pro Ile530 535 540Gln Val Asn Pro
Val Asn Asp Pro Pro His Ile Ile Phe Pro His Gly545 550 555 560Ser
Leu Met Val Ile Leu Glu His Thr Gln Lys Pro Leu Gly Pro Glu565 570
575Val Phe Gln Ala Tyr Asp Pro Asp Ser Ala Cys Glu Gly Leu Thr
Phe580 585 590Gln Val Leu Gly Thr Ser Ser Gly Leu Pro Val Glu Arg
Arg Asp Gln595 600 605Pro Gly Glu Pro Ala Thr Glu Phe Ser Cys Arg
Glu Leu Glu Ala Gly610 615 620Ser Leu Val Tyr Val His Arg Gly Gly
Pro Ala Gln Asp Leu Thr Phe625 630 635 640Arg Val Ser Asp Gly Leu
Gln Ala Ser Pro Pro Ala Thr Leu Lys Val645 650 655Val Ala Ile Arg
Pro Ala Ile Gln Ile His Arg Ser Thr Gly Leu Arg660 665 670Leu Ala
Gln Gly Ser Ala Met Pro Ile Leu Pro Ala Asn Leu Ser Val675 680
685Glu Thr Asn Ala Val Gly Gln Asp Val Ser Val Leu Phe Arg Val
Thr690 695 700Gly Ala Leu Gln Phe Gly Glu Leu Gln Lys Gln Gly Ala
Gly Gly Val705 710 715 720Glu Gly Ala Glu Trp Trp Ala Thr Gln Ala
Phe His Gln Arg Asp Val725 730 735Glu Gln Gly Arg Val Arg Tyr Leu
Ser Thr Asp Pro Gln His His Ala740 745 750Tyr Asp Thr Val Glu Asn
Leu Ala Leu Glu Val Gln Val Gly Gln Glu755 760 765Ile Leu Ser Asn
Leu Ser Phe Pro Val Thr Ile Gln Arg Ala Thr Val770 775 780Trp Met
Leu Arg Leu Glu Pro Leu His Thr Gln Asn Thr Gln Gln Glu785 790 795
800Thr Leu Thr Thr Ala His Leu Glu Ala Thr Leu Glu Glu Ala Gly
Pro805 810 815Ser Pro Pro Thr Phe His Tyr Glu Val Val Gln Ala Pro
Arg Lys Gly820 825 830Asn Leu Gln Leu Gln Gly Thr Arg Leu Ser Asp
Gly Gln Gly Phe Thr835 840 845Gln Asp Asp Ile Gln Ala Gly Arg Val
Thr Tyr Gly Ala Thr Ala Arg850 855 860Ala Ser Glu Ala Val Glu Asp
Thr Phe Arg Phe Arg Val Thr Ala Pro865 870 875 880Pro Tyr Phe Ser
Pro Leu Tyr Thr Phe Pro Ile His Ile Gly Gly Asp885 890 895Pro Asp
Ala Pro Val Leu Thr Asn Val Leu Leu Val Val Pro Glu Gly900 905
910Gly Glu Gly Val Leu Ser Ala Asp His Leu Phe Val Lys Ser Leu
Asn915 920 925Ser Ala Ser Tyr Leu Tyr Glu Val Met Glu Arg Pro Arg
His Gly Arg930 935 940Leu Ala Trp Arg Gly Thr Gln Asp Lys Thr Thr
Met Val Thr Ser Phe945 950 955 960Thr Asn Glu Asp Leu Leu Arg Gly
Arg Leu Val Tyr Gln His Asp Asp965 970 975Ser Glu Thr Thr Glu Asp
Asp Ile Pro Phe Val Ala Thr Arg Gln Gly980 985 990Glu Ser Ser Gly
Asp Met Ala Trp Glu Glu Val Arg Gly Val Phe Arg995 1000 1005Val Ala
Ile Gln Pro Val Asn Asp His Ala Pro Val Gln Thr Ile Ser1010 1015
1020Arg Ile Phe His Val Ala Arg Gly Gly Arg Arg Leu Leu Thr Thr
Asp1025 1030 1035 1040Asp Val Ala Phe Ser Asp Ala Asp Ser Gly Phe
Ala Asp Ala Gln Leu1045 1050 1055Val Leu Thr Arg Lys Asp Leu Leu
Phe Gly Ser Ile Val Ala Val Asp1060 1065 1070Glu Pro Thr Arg Pro
Ile Tyr Arg Phe Thr Gln Glu Asp Leu Arg Lys1075 1080 1085Arg Arg
Val Leu Phe Val His Ser Gly Ala Asp Arg Gly Trp Ile Gln1090 1095
1100Leu Gln Val Ser Asp Gly Gln His Gln Ala Thr Ala Leu Leu Glu
Val1105 1110 1115 1120Gln Ala Ser Glu Pro Tyr Leu Arg Val Ala Asn
Gly Ser Ser Leu Val1125 1130 1135Val Pro Gln Gly Gly Gln Gly Thr
Ile Asp Thr Ala Val Leu His Leu1140 1145 1150Asp Thr Asn Leu Asp
Ile Arg Ser Gly Asp Glu Val His Tyr His Val1155 1160 1165Thr Ala
Gly Pro Arg Trp Gly Gln Leu Val Arg Ala Gly Gln Pro Ala1170 1175
1180Thr Ala Phe Ser Gln Gln Asp Leu Leu Asp Gly Ala Val Leu Tyr
Ser1185 1190 1195 1200His Asn Gly Ser Leu Ser Pro Arg Asp Thr Met
Ala Phe Ser Val Glu1205 1210 1215Ala Gly Pro Val His Thr Asp Ala
Thr Leu Gln Val Thr Ile Ala Leu1220 1225 1230Glu Gly Pro Leu Ala
Pro Leu Lys Leu Val Arg His Lys Lys Ile Tyr1235 1240 1245Val Phe
Gln Gly Glu Ala Ala Glu Ile Arg Arg Asp Gln Leu Glu Ala1250 1255
1260Ala Gln Glu Ala Val Pro Pro Ala Asp Ile Val Phe Ser Val Lys
Ser1265 1270 1275 1280Pro Pro Ser Ala Gly Tyr Leu Val Met Val Ser
Arg Gly Ala Leu Ala1285 1290 1295Asp Glu Pro Pro Ser Leu Asp Pro
Val Gln Ser Phe Ser Gln Glu Ala1300 1305 1310Val Asp Thr Gly Arg
Val Leu Tyr Leu His Ser Arg Pro Glu Ala Trp1315 1320 1325Ser Asp
Ala Phe Ser Leu Asp Val Ala Ser Gly Leu Gly Ala Pro Leu1330 1335
1340Glu Gly Val Leu Val Glu Leu Glu Val Leu Pro Ala Ala Ile Pro
Leu1345 1350 1355 1360Glu Ala Gln Asn Phe Ser Val Pro Glu Gly Gly
Ser Leu Thr Leu Ala1365 1370 1375Pro Pro Leu Leu Arg Val Ser Gly
Pro Tyr Phe Pro Thr Leu Leu Gly1380 1385 1390Leu Ser Leu Gln Val
Leu Glu Pro Pro Gln His Gly Ala Leu Gln Lys1395 1400 1405Glu Asp
Gly Pro Gln Ala Arg Thr Leu Ser Ala Phe Ser Trp Arg Met1410 1415
1420Val Glu Glu Gln Leu Ile Arg Tyr Val His Asp Gly Ser Glu Thr
Leu1425 1430 1435 1440Thr Asp Ser Phe Val Leu Met Ala Asn Ala Ser
Glu Met Asp Arg Gln1445 1450 1455Ser His Pro Val Ala Phe Thr Val
Thr Val Leu Pro Val Asn Asp Gln1460 1465 1470Pro Pro Ile Leu Thr
Thr Asn Thr Gly Leu Gln Met Trp Glu Gly Ala1475 1480 1485Thr Ala
Pro Ile Pro Ala Glu Ala Leu Arg Ser Thr Asp Gly Asp Ser1490 1495
1500Gly Ser Glu Asp Leu Val Tyr Thr Ile Glu Gln Pro Ser Asn Gly
Arg1505 1510 1515 1520Val Val Leu Arg Gly Ala Pro Gly Thr Glu Val
Arg Ser Phe Thr Gln1525 1530 1535Ala Gln Leu Asp Gly Gly Leu Val
Leu Phe Ser His Arg Gly Thr Leu1540 1545 1550Asp Gly Gly Phe Arg
Phe Arg Leu Ser Asp Gly Glu His Thr Ser Pro1555 1560 1565Gly His
Phe Phe Arg Val Thr Ala Gln Lys Gln Val Leu Leu Ser Leu1570 1575
1580Lys Gly Ser Gln Thr Leu Thr Val Cys Pro Gly Ser Val Gln Pro
Leu1585 1590 1595 1600Ser Ser Gln Thr Leu Arg Ala Ser Ser Ser Ala
Gly Thr Asp Pro Gln1605 1610 1615Leu Leu Leu Tyr Arg Val Val Arg
Gly Pro Gln Leu Gly Arg Leu Phe1620 1625 1630His Ala Gln Gln Asp
Ser Thr Gly Glu Ala Leu Val Asn Phe Thr Gln1635 1640 1645Ala Glu
Val Tyr Ala Gly Asn Ile Leu Tyr Glu His Glu Met Pro Pro1650 1655
1660Glu Pro Phe Trp Glu Ala His Asp Thr Leu Glu Leu Gln Leu Ser
Ser1665 1670 1675 1680Pro Pro Ala Arg Asp Val Ala Ala Thr Leu Ala
Val Ala Val Ser Phe1685 1690 1695Glu Ala Ala Cys Pro Gln Arg Pro
Ser His Leu Trp Lys Asn Lys Gly1700 1705 1710Leu Trp Val Pro Glu
Gly Gln Arg Ala Arg Ile Thr Val Ala Ala Leu1715 1720 1725Asp Ala
Ser Asn Leu Leu Ala Ser Val Pro Ser Pro Gln Arg Ser Glu1730 1735
1740His Asp Val Leu Phe Gln Val Thr Gln Phe Pro Ser Arg Gly Gln
Leu1745 1750 1755 1760Leu Val Ser Glu Glu Pro Leu His Ala Gly Gln
Pro His Phe Leu Gln1765 1770 1775Ser Gln Leu Ala Ala Gly Gln Leu
Val Tyr Ala His Gly Gly Gly Gly1780 1785 1790Thr Gln Gln Asp Gly
Phe His Phe Arg Ala His Leu Gln Gly Pro Ala1795 1800 1805Gly Ala
Ser Val Ala Gly Pro Gln Thr Ser Glu Ala Phe Ala Ile Thr1810 1815
1820Val Arg Asp Val Asn Glu Arg Pro Pro Gln Pro Gln Ala Ser Val
Pro1825 1830 1835 1840Leu Arg Leu Thr Arg Gly Ser Arg Ala Pro Ile
Ser Arg Ala Gln Leu1845 1850 1855Ser Val Val Asp Pro Asp Ser Ala
Pro Gly Glu Ile Glu Tyr Glu Val1860 1865 1870Gln Arg Ala Pro His
Asn Gly Phe Leu Ser Leu Val Gly Gly Gly Leu1875 1880 1885Gly Pro
Val Thr Arg Phe Thr Gln Ala Asp Val Asp Ser Gly Arg Leu1890 1895
1900Ala Phe Val Ala Asn Gly Ser Ser Val Ala Gly Ile Phe Gln Leu
Ser1905 1910 1915 1920Met Ser Asp Gly Ala Ser Pro Pro Leu Pro Met
Ser Leu Ala Val Asp1925 1930 1935Ile Leu Pro Ser Ala Ile Glu Val
Gln Leu Arg Ala Pro Leu Glu Val1940 1945 1950Pro Gln Ala Leu Gly
Arg Ser Ser Leu Ser Gln Gln Gln Leu Arg Val1955 1960 1965Val Ser
Asp Arg Glu Glu Pro Glu Ala Ala Tyr Arg Leu Ile Gln Gly1970 1975
1980Pro Gln Tyr Gly His Leu Leu Val Gly Gly Arg Pro Thr Ser Ala
Phe1985 1990 1995 2000Ser Gln Phe Gln Ile Asp Gln Gly Glu Val Val
Phe Ala Phe Thr Asn2005 2010 2015Phe Ser Ser Ser His Asp His Phe
Arg Val Leu Ala Leu Ala Arg Gly2020 2025 2030Val Asn Ala Ser Ala
Val Val Asn Val Thr Val Arg Ala Leu Leu His2035 2040 2045Val Trp
Ala Gly Gly Pro Trp Pro Gln Gly Ala Thr Leu Arg Leu Asp2050 2055
2060Pro Thr Val Leu Asp Ala Gly Glu Leu Ala Asn Arg Thr Gly Ser
Val2065 2070 2075 2080Pro Arg Phe Arg Leu Leu Glu Gly Pro Arg His
Gly Arg Val Val Arg2085 2090 2095Val Pro Arg Ala Arg Thr Glu Pro
Gly Gly Ser Gln Leu Val Glu Gln2100 2105 2110Phe Thr Gln Gln Asp
Leu Glu Asp Gly Arg Leu Gly Leu Glu Val Gly2115 2120 2125Arg Pro
Glu Gly Arg Ala Pro Gly Pro Ala Gly Asp Ser Leu Thr Leu2130 2135
2140Glu Leu Trp Ala Gln Gly Val Pro Pro Ala Val Ala Ser Leu Asp
Phe2145 2150 2155 2160Ala Thr Glu Pro Tyr Asn Ala Ala Arg Pro Tyr
Ser Val Ala Leu Leu2165 2170 2175Ser Val Pro Glu Ala Ala Arg Thr
Glu Ala Gly Lys Pro Glu Ser Ser2180 2185 2190Thr Pro Thr Gly Glu
Pro Gly Pro Met Ala Ser Ser Pro Glu Pro Ala2195 2200 2205Val Ala
Lys Gly Gly Phe Leu Ser Phe Leu Glu Ala Asn Met Phe Ser2210 2215
2220Val Ile Ile Pro Met Cys Leu Val Leu Leu Leu Leu Ala Leu Ile
Leu2225 2230 2235 2240Pro Leu Leu Phe Tyr Leu Arg Lys Arg Asn Lys
Thr Gly Lys His Asp2245 2250 2255Val Gln Val Leu Thr Ala Lys Pro
Arg Asn Gly Leu Ala Gly Asp Thr2260 2265 2270Glu Thr Phe Arg Lys
Val Glu Pro Gly Gln Ala Ile Pro Leu Thr Ala2275 2280 2285Val Pro
Gly Gln Gly Pro Pro Pro Gly Gly Gln Pro Asp Pro Glu Leu2290 2295
2300Leu Gln Phe Cys Arg Thr Pro Asn Pro Ala Leu Lys Asn Gly Gln
Tyr2305 2310 2315 2320Trp Val27011DNAHomo sapiens 2atgcagtccg
gccgcggccc cccacttcca gcccccggcc tggccttggc tttgaccctg 60actatgttgg
ccagacttgc atccgcggct tccttcttcg gtgagaacca cctggaggtg
120cctgtggcca cggctctgac cgacatagac ctgcagctgc agttctccac
gtcccagccc 180gaagccctcc ttctcctggc agcaggccca gctgaccacc
tcctgctgca gctctactct 240ggacgcctgc aggtcagact tgttctgggc
caggaggagc tgaggctgca gactccagca 300gagacgctgc tgagtgactc
catcccccac actgtggtgc tgactgtcgt agagggctgg 360gccacgttgt
cagtcgatgg gtttctgaac gcctcctcag cagtcccagg agccccccta
420gaggtcccct atgggctctt tgttgggggc actgggaccc ttggcctgcc
ctacctgagg 480ggaaccagcc gacccctgag gggttgcctc catgcagcca
ccctcaatgg ccgcagcctc 540ctccggcctc tgacccccga tgtgcatgag
ggctgtgctg aagagttttc tgccagtgat 600gatgtggccc tgggcttctc
tgggccccac tctctggctg ccttccctgc ctggggcact 660caggacgaag
gaaccctaga gtttacactc accacacaga gccggcaggc acccttggcc
720ttccaggcag ggggccggcg tggggacttc atctatgtgg acatatttga
gggccacctg 780cgggccgtgg tggagaaggg ccagggtacc gtattgctcc
acaacagtgt gcctgtggcc 840gatgggcagc cccatgaggt cagtgtccac
atcaatgctc accggctgga aatctccgtg 900gaccagtacc ctacgcatac
ttcgaaccga ggagtcctca gctacctgga gccacggggc 960agtctccttc
tcggggggct ggatgcagag gcctctcgtc acctccagga acaccgcctg
1020ggcctgacac cagaggccac caatgcctcc ctgctgggct gcatggaaga
cctcagtgtc 1080aatggccaga ggcgggggct gcgggaagct ttgctgacgc
gcaacatggc agccggctgc 1140aggctggagg aggaggagta tgaggacgat
gcctatggac attatgaagc tttctccacc 1200ctggcccctg aggcttggcc
agccatggag ctgcctgagc catgcgtgcc tgagccaggg 1260ctgcctcctg
tctttgccaa tttcacccag ctgctgacta tcagcccact ggtggtggcc
1320gaggggggca cagcctggct tgagtggagg catgtgcagc ccacgctgga
cctgatggag 1380gctgagctgc gcaaatccca ggtgctgttc agcgtgaccc
gaggggcacg ccatggcgag 1440ctcgagctgg acatcccggg agcccaggca
cgaaaaatgt tcaccctcct ggacgtggtg 1500aaccgcaagg cccgcttcat
ccacgatggc tctgaggaca cctccgacca gctggtgctg 1560gaggtgtcgg
tgacggctcg ggtgcccatg ccctcatgcc ttcggagggg ccaaacatac
1620ctcctgccca tccaggtcaa ccctgtcaat gacccacccc acatcatctt
cccacatggc 1680agcctcatgg tgatcctgga acacacgcag aagccgctgg
ggcctgaggt tttccaggcc 1740tatgacccgg actctgcctg
tgagggcctc accttccagg tccttggcac ctcctctggc 1800ctccccgtgg
agcgccgaga ccagcctggg gagccggcga ccgagttctc ctgccgggag
1860ttggaggccg gcagcctagt ctatgtccac cgcggtggtc ctgcacagga
cttgacgttc 1920cgggtcagcg atggactgca ggccagcccc ccggccacgc
tgaaggtggt ggccatccgg 1980ccggccatac agatccaccg cagcacaggg
ttgcgactgg cccaaggctc tgccatgccc 2040atcttgcccg ccaacctgtc
ggtggagacc aatgccgtgg ggcaggatgt gagcgtgctg 2100ttccgcgtca
ctggggccct gcagtttggg gagctgcaga agcagggggc aggtggggtg
2160gagggtgctg agtggtgggc cacacaggcg ttccaccagc gggatgtgga
gcagggccgc 2220gtgaggtacc tgagcactga cccacagcac cacgcttacg
acaccgtgga gaacctggcc 2280ctggaggtgc aggtgggcca ggagatcctg
agcaatctgt ccttcccagt gaccatccag 2340agagccactg tgtggatgct
gcggctggag ccactgcaca ctcagaacac ccagcaggag 2400accctcacca
cagcccacct ggaggccacc ctggaggagg caggcccaag ccccccaacc
2460ttccattatg aggtggttca ggctcccagg aaaggcaacc ttcaactaca
gggcacaagg 2520ctgtcagatg gccagggctt cacccaggat gacatacagg
ctggccgggt gacctatggg 2580gccacagcac gtgcctcaga ggcagtcgag
gacaccttcc gtttccgtgt cacagctcca 2640ccatatttct ccccactcta
taccttcccc atccacattg gtggtgaccc agatgcgcct 2700gtcctcacca
atgtcctcct cgtggtgcct gagggtggtg agggtgtcct ctctgctgac
2760cacctctttg tcaagagtct caacagtgcc agctacctct atgaggtcat
ggagcggccc 2820cgccatggga ggttggcttg gcgtgggaca caggacaaga
ccactatggt gacatccttc 2880accaatgaag acctgttgcg tggccggctg
gtctaccagc atgatgactc cgagaccaca 2940gaagatgata tcccatttgt
tgctacccgc cagggcgaga gcagtggtga catggcctgg 3000gaggaggtac
ggggtgtctt ccgagtggcc atccagcccg tgaatgacca cgcccctgtg
3060cagaccatca gccggatctt ccatgtggcc cggggtgggc ggcggctgct
gactacagac 3120gacgtggcct tcagcgatgc tgactcgggc tttgctgacg
cccagctggt gcttacccgc 3180aaggacctcc tctttggcag tatcgtggcc
gtagatgagc ccacgcggcc catctaccgc 3240ttcacccagg aggacctcag
gaagaggcga gtactgttcg tgcactcagg ggctgaccgt 3300ggctggatcc
agctgcaggt gtccgacggg caacaccagg ccactgcgct gctggaggtg
3360caggcctcgg aaccctacct ccgtgtggcc aacggctcca gccttgtggt
ccctcaaggg 3420ggccagggca ccatcgacac ggccgtgctc cacctggaca
ccaacctcga catccgcagt 3480ggggatgagg tccactacca cgtcacagct
ggccctcgct ggggacagct agtccgggct 3540ggtcagccag ccacagcctt
ctcccagcag gacctgctgg atggggccgt tctctatagc 3600cacaatggca
gcctcagccc ccgcgacacc atggccttct ccgtggaagc agggccagtg
3660cacacggatg ccaccctaca agtgaccatt gccctagagg gcccactggc
cccactgaag 3720ctggtccggc acaagaagat ctacgtcttc cagggagagg
cagctgagat cagaagggac 3780cagctggagg cagcccagga ggcagtgcca
cctgcagaca tcgtattctc agtgaagagc 3840ccaccgagtg ccggctacct
ggtgatggtg tcgcgtggcg ccttggcaga tgagccaccc 3900agcctggacc
ctgtgcagag cttctcccag gaggcagtgg acacaggcag ggtcctgtac
3960ctgcactccc gccctgaggc ctggagcgat gccttctcgc tggatgtggc
ctcaggcctg 4020ggtgctcccc tcgagggcgt ccttgtggag ctggaggtgc
tgcccgctgc catcccacta 4080gaggcgcaaa acttcagcgt ccctgagggt
ggcagcctca ccctggcccc tccactgctc 4140cgtgtctccg ggccctactt
ccccactctc ctgggcctca gcctgcaggt gctggagcca 4200ccccagcatg
gagccctgca gaaggaggac ggacctcaag ccaggaccct cagcgccttc
4260tcctggagaa tggtggaaga gcagctgatc cgctacgtgc atgacgggag
cgagacactg 4320acagacagtt ttgtcctgat ggctaatgcc tccgagatgg
atcgccagag ccatcctgtg 4380gccttcactg tcactgtcct gcctgtcaat
gaccaacccc ccatcctcac tacaaacaca 4440ggcctgcaga tgtgggaggg
ggccactgcg cccatccctg cggaggctct gaggagcacg 4500gacggcgact
ctgggtctga ggatctggtc tacaccatcg agcagcccag caacgggcgg
4560gtagtgctgc ggggggcgcc gggcactgag gtgcgcagct tcacgcaggc
ccagctggac 4620ggcgggctcg tgctgttctc acacagagga accctggatg
gaggcttccg cttccgcctc 4680tctgacggcg agcacacttc ccccggacac
ttcttccgag tgacggccca gaagcaagtg 4740ctcctctcgc tgaagggcag
ccagacactg actgtctgcc cagggtccgt ccagccactc 4800agcagtcaga
ccctcagggc cagctccagc gcaggcactg acccccagct cctgctctac
4860cgtgtggtgc ggggccccca gctaggccgg ctgttccacg cccagcagga
cagcacaggg 4920gaggccctgg tgaacttcac tcaggcagag gtctacgctg
ggaatattct gtatgagcat 4980gagatgcccc ccgagccctt ttgggaggcc
catgataccc tagagctcca gctgtcctcg 5040ccgcctgccc gggacgtggc
cgccaccctt gctgtggctg tgtcttttga ggctgcctgt 5100ccccagcgcc
ccagccacct ctggaagaac aaaggtctct gggtccccga gggccagcgg
5160gccaggatca ccgtggctgc tctggatgcc tccaatctct tggccagcgt
tccatcaccc 5220cagcgctcag agcatgatgt gctcttccag gtcacacagt
tccccagccg gggccagctg 5280ttggtgtccg aggagcccct ccatgctggg
cagccccact tcctgcagtc ccagctggct 5340gcagggcagc tagtgtatgc
ccacggcggt gggggcaccc agcaggatgg cttccacttt 5400cgtgcccacc
tccaggggcc agcaggggcc tccgtggctg gaccccaaac ctcagaggcc
5460tttgccatca cggtgaggga tgtaaatgag cggccccctc agccacaggc
ctctgtccca 5520ctccggctca cccgaggctc tcgtgccccc atctcccggg
cccagctgag tgtggtggac 5580ccagactcag ctcctgggga gattgagtac
gaggtccagc gggcacccca caacggcttc 5640ctcagcctgg tgggtggtgg
cctggggccc gtgacccgct tcacgcaagc cgatgtggat 5700tcagggcggc
tggccttcgt ggccaacggg agcagcgtgg caggcatctt ccagctgagc
5760atgtctgatg gggccagccc acccctgccc atgtccctgg ctgtggacat
cctaccatcc 5820gccatcgagg tgcagctgcg ggcacccctg gaggtgcccc
aagctttggg gcgctcctca 5880ctgagccagc agcagctccg ggtggtttca
gatcgggagg agccagaggc agcataccgc 5940ctcatccagg gaccccagta
tgggcatctc ctggtgggcg ggcggcccac ctcggccttc 6000agccaattcc
agatagacca gggcgaggtg gtctttgcct tcaccaactt ctcctcctct
6060catgaccact tcagagtcct ggcactggct aggggtgtca atgcatcagc
cgtagtgaac 6120gtcactgtga gggctctgct gcatgtgtgg gcaggtgggc
catggcccca gggtgccacc 6180ctgcgcctgg accccaccgt cctagatgct
ggcgagctgg ccaaccgcac aggcagtgtg 6240ccgcgcttcc gcctcctgga
gggaccccgg catggccgcg tggtccgcgt gccccgagcc 6300aggacggagc
ccgggggcag ccagctggtg gagcagttca ctcagcagga ccttgaggac
6360gggaggctgg ggctggaggt gggcaggcca gaggggaggg cccccggccc
cgcaggtgac 6420agtctcactc tggagctgtg ggcacagggc gtcccgcctg
ctgtggcctc cctggacttt 6480gccactgagc cttacaatgc tgcccggccc
tacagcgtgg ccctgctcag tgtccccgag 6540gccgcccgga cggaagcagg
gaagccagag agcagcaccc ccacaggcga gccaggcccc 6600atggcatcca
gccctgagcc cgctgtggcc aagggaggct tcctgagctt ccttgaggcc
6660aacatgttca gcgtcatcat ccccatgtgc ctggtacttc tgctcctggc
gctcatcctg 6720cccctgctct tctacctccg aaaacgcaac aagacgggca
agcatgacgt ccaggtcctg 6780actgccaagc cccgcaacgg cctggctggt
gacaccgaga cctttcgcaa ggtggagcca 6840ggccaggcca tcccgctcac
agctgtgcct ggccaggggc cccctccagg aggccagcct 6900gacccagagc
tgctgcagtt ctgccggaca cccaaccctg cccttaagaa tggccagtac
6960tgggtgtgag gcctggcctg ggcccagatg ctgatcgggc cagggacagg c
7011342PRTArtificial SequenceDescription of Artificial Sequnece
T-cell stimulatory peptide 3Leu Ala Gln Gly Ser Ala Met Pro Ile Leu
Pro Ala Asn Leu Ser Val 1 5 10 15Glu Thr Asn Ala Val Gly Gln Asp
Val Ser Val Leu Phe Arg Val Thr20 25 30Gly Ala Leu Gln Phe Gly Glu
Leu Gln Lys35 40416PRTArtificial SequenceDescription of Artificial
Sequnece T-cell stimulatory peptide 4Leu Ala Gln Gly Ser Ala Met
Pro Ile Leu Pro Ala Asn Leu Ser Val 1 5 10 15516PRTArtificial
SequenceDescription of Artificial Sequnece T-cell stimulatory
peptide 5Ser Ala Met Pro Ile Leu Pro Ala Asn Leu Ser Val Glu Thr
Asn Ala 1 5 10 15616PRTArtificial SequenceDescription of Artificial
Sequnece T-cell stimulatory peptide 6Ile Leu Pro Ala Asn Leu Ser
Val Glu Thr Asn Ala Val Gly Gln Asp 1 5 10 15716PRTArtificial
SequenceDescription of Artificial Sequnece T-cell stimulatory
peptide 7Asn Leu Ser Val Glu Thr Asn Ala Val Gly Gln Asp Val Ser
Val Leu 1 5 10 15816PRTArtificial SequenceDescription of Artificial
Sequnece T-cell stimulatory peptide 8Glu Thr Asn Ala Val Gly Gln
Asp Val Ser Val Leu Phe Arg Val Thr 1 5 10 15916PRTArtificial
SequenceDescription of Artificial Sequnece T-cell stimulatory
peptide 9Val Gly Gln Asp Val Ser Val Leu Phe Arg Val Thr Gly Ala
Leu Gln 1 5 10 151016PRTArtificial SequenceDescription of
Artificial Sequnece T-cell stimulatory peptide 10Val Ser Val Leu
Phe Arg Val Thr Gly Ala Leu Gln Phe Gly Glu Leu 1 5 10
151114PRTArtificial SequenceDescription of Artificial Sequnece
T-cell stimulatory peptide 11Phe Arg Val Thr Gly Ala Leu Gln Phe
Gly Glu Leu Gln Lys 1 5 101216PRTArtificial SequenceDescription of
Artificial Sequence Peptide derived from MCSP 12Ser Gln Val Leu Phe
Ser Val Thr Arg Gly Ala His Tyr Gly Glu Leu 1 5 10
151316PRTArtificial SequenceDescription of Artificial Sequence
Peptide derived from MCSP 13Val Arg Tyr Leu Ser Thr Asp Pro Gln His
His Ala Tyr Asp Thr Val 1 5 10 151416PRTArtificial
SequenceDescription of Artificial Sequence Peptide derived from
MCSP 14Gly Glu Ala Leu Val Asn Phe Thr Gln Ala Glu Val Tyr Ala Gly
Asn 1 5 10 151516PRTArtificial SequenceDescription of Artificial
Sequence Peptide derived from MCSP 15Pro His Glu Val Ser Val His
Ile Asn Ala His Arg Leu Glu Ile Ser 1 5 10 151627DNAArtificial
SequenceDescription of Artificial Sequence forward primer for MCSP
16ggggatccca tccggccggc catacag 271731DNAArtificial
SequenceDescription of Artificial Sequence reverse primer for MCSP
17ggggatcctc accgctggtg gaacgcctgt g 311823DNAArtificial
SequenceDescription of Artificial Sequence sense primer for hu-Ii
18tttccatgga tgaccagcgc gac 231930DNAArtificial SequenceDescription
of Artificial Sequence antisense primer for hu-Ii 19tttggatccg
gaagcttcat gcgcaggttc 3020474DNAArtificial SequenceDescription of
Artificial Sequence DNA sequence showing the hu-Ii- MCSP fusion
20atggacctta tctccaacaa tgagcaactg cccatgctgg gccggcgccc tggggccccg
60gagagcaagt gcagccgcgg agccctgtac acaggctttt ccatcctggt gactctgctc
120ctcgctggcc aggccaccac cgcctacttc ctgtaccagc agcagggccg
gctggacaaa 180ctgacagtca cctcccagaa cctgcagctg gagaacctgc
gcatgaagct tcccaaggat 240cccatccggc cggccataca gatccaccgc
agcacagggt tgcgactggc ccaaggctct 300gccatgccca tcttgcccgc
caacctgtcg gtggagacca atgccgtggg gcaggatgtg 360agcgtgctgt
tccgcgtcac tggggccctg cagtttgggg agctgcagaa gcagggggca
420ggtggggtgg agggtgctga gtggtgggcc acacaggcgt tccaccagcg gtga
47421158PRTArtificial SequenceDescription of Artificial Sequence
hu-Ii- MCSP fusion peptide 21Met Asp Leu Ile Ser Asn Asn Glu Gln
Leu Pro Met Leu Gly Arg Arg 1 5 10 15Pro Gly Ala Pro Glu Ser Lys
Cys Ser Arg Gly Ala Leu Tyr Thr Gly20 25 30Phe Ser Ile Leu Val Thr
Leu Leu Leu Ala Gly Gln Ala Thr Thr Ala35 40 45Tyr Phe Leu Tyr Gln
Gln Gln Gly Arg Leu Asp Lys Leu Thr Val Thr50 55 60Ser Gln Asn Leu
Gln Leu Glu Asn Leu Arg Met Lys Leu Pro Lys Asp65 70 75 80Pro Ile
Arg Pro Ala Ile Gln Ile His Arg Ser Thr Gly Leu Arg Leu85 90 95Ala
Gln Gly Ser Ala Met Pro Ile Leu Pro Ala Asn Leu Ser Val Glu100 105
110Thr Asn Ala Val Gly Gln Asp Val Ser Val Leu Phe Arg Val Thr
Gly115 120 125Ala Leu Gln Phe Gly Glu Leu Gln Lys Gln Gly Ala Gly
Gly Val Glu130 135 140Gly Ala Glu Trp Trp Ala Thr Gln Ala Phe His
Gln Arg Glx145 150 155229PRTArtificial SequenceDescription of
Artificial Sequence truncated peptide 22Leu Phe Arg Val Thr Gly Ala
Leu Gln1 52310PRTArtificial SequenceDescription of Artificial
Sequence truncated peptide 23Val Leu Phe Arg Val Thr Gly Ala Leu
Gln 1 5 102411PRTArtificial SequenceDescription of Artificial
Sequence truncated peptide 24Ser Val Leu Phe Arg Val Thr Gly Ala
Leu Gln 1 5 102512PRTArtificial SequenceDescription of Artificial
Sequence truncated peptide 25Val Ser Val Leu Phe Arg Val Thr Gly
Ala Leu Gln1 5 102613PRTArtificial SequenceDescription of
Artificial Sequence truncated peptide 26Asp Val Ser Val Leu Phe Arg
Val Thr Gly Ala Leu Gln1 5 102714PRTArtificial SequenceDescription
of Artificial Sequence truncated peptide 27Gln Asp Val Ser Val Leu
Phe Arg Val Thr Gly Ala Leu Gln1 5 102815PRTArtificial
SequenceDescription of Artificial Sequence truncated peptide 28Gly
Gln Asp Val Ser Val Leu Phe Arg Val Thr Gly Ala Leu Gln1 5 10
152915PRTArtificial SequenceDescription of Artificial Sequence
truncated peptide 29Val Gly Gln Asp Val Ser Val Leu Phe Arg Val Thr
Gly Ala Leu1 5 10 153014PRTArtificial SequenceDescription of
Artificial Sequence truncated peptide 30Val Gly Gln Asp Val Ser Val
Leu Phe Arg Val Thr Gly Ala1 5 103113PRTArtificial
SequenceDescription of Artificial Sequence truncated peptide 31Val
Gly Gln Asp Val Ser Val Leu Phe Arg Val Thr Gly1 5
103212PRTArtificial SequenceDescription of Artificial Sequence
truncated peptide 32Val Gly Gln Asp Val Ser Val Leu Phe Arg Val
Thr1 5 103311PRTArtificial SequenceDescription of Artificial
Sequence truncated peptide 33Val Gly Gln Asp Val Ser Val Leu Phe
Arg Val1 5 103410PRTArtificial SequenceDescription of Artificial
Sequence truncated peptide 34Val Gly Gln Asp Val Ser Val Leu Phe
Arg1 5 10359PRTArtificial SequenceDescription of Artificial
Sequence truncated peptide 35Val Gly Gln Asp Val Ser Val Leu Phe1
53631PRTArtificial SequenceDescription of Artificial Sequence
fragment of MCSP 36Pro Pro Ala Asp Ile Val Phe Ser Val Lys Ser Pro
Pro Ser Ala Gly1 5 10 15Tyr Leu Val Met Val Ser Arg Gly Ala Leu Ala
Asp Glu Pro Pro20 25 303711PRTArtificial SequenceDescription of
Artificial Sequence synthetic construct 37Pro Pro Ser Ala Gly Tyr
Leu Val Met Val Ser1 5 103812PRTArtificial SequenceDescription of
Artificial Sequence fragment of MCSP 38Pro Pro Ser Ala Gly Tyr Leu
Val Met Val Ser Arg1 5 103913PRTArtificial SequenceDescription of
Artificial Sequence fragment of MCSP 39Pro Pro Ser Ala Gly Tyr Leu
Val Met Val Ser Arg Gly1 5 104014PRTArtificial SequenceDescription
of Artificial Sequence fragment of MCSP 40Pro Pro Ser Ala Gly Tyr
Leu Val Met Val Ser Arg Gly Ala1 5 104115PRTArtificial
SequenceDescription of Artificial Sequence fragment of MCSP 41Pro
Pro Ser Ala Gly Tyr Leu Val Met Val Ser Arg Gly Ala Leu1 5 10
154216PRTArtificial SequenceDescription of Artificial Sequence
fragment of MCSP 42Pro Pro Ser Ala Gly Tyr Leu Val Met Val Ser Arg
Gly Ala Leu Ala1 5 10 154315PRTArtificial SequenceDescription of
Artificial Sequence fragment of MCSP 43Pro Ser Ala Gly Tyr Leu Val
Met Val Ser Arg Gly Ala Leu Ala1 5 10 154414PRTArtificial
SequenceDescription of Artificial Sequence fragment of MCSP 44Ser
Ala Gly Tyr Leu Val Met Val Ser Arg Gly Ala Leu Ala1 5
104513PRTArtificial SequenceDescription of Artificial Sequence
fragment of MCSP 45Ala Gly Tyr Leu Val Met Val Ser Arg Gly Ala Leu
Ala1 5 104612PRTArtificial SequenceDescription of Artificial
Sequence fragment of MCSP 46Gly Tyr Leu Val Met Val Ser Arg Gly Ala
Leu Ala1 5 104711PRTArtificial SequenceDescription of Artificial
Sequence fragment of MCSP 47Tyr Leu Val Met Val Ser Arg Gly Ala Leu
Ala1 5 104816PRTArtificial SequenceDescription of Artificial
Sequence fragment of MCSP 48Pro Pro Ala Asp Ile Val Phe Ser Val Lys
Ser Pro Pro Ser Ala Gly1 5 10 154915PRTArtificial
SequenceDescription of Artificial Sequence fragment of MCSP 49Ile
Val Phe Ser Val Lys Ser Pro Pro Ser Ala Gly Tyr Leu Val1 5 10
155016PRTArtificial SequenceDescription of Artificial Sequence
fragment of MCSP 50Ser Val Lys Ser Pro Pro Ser Ala Gly Tyr Leu Val
Met Val Ser Arg1 5 10 155116PRTArtificial SequenceDescription of
Artificial Sequence fragment of MCSP 51Gly Tyr Leu Val Met Val Ser
Arg Gly Ala Leu Ala Asp Glu Pro Pro1 5 10 155211PRTArtificial
SequenceDescription of Artificial Sequence fragment of MCSP 52Tyr
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg1 5 105311PRTArtificial
SequenceDescription of Artificial Sequence HIV 53Tyr Ala Arg Ala
Ala Ala Arg Gln Ala Arg Ala1 5 105442PRTArtificial
SequenceDescription of Artificial Sequence fusion peptide 54Tyr Ala
Arg Ala Ala Ala Arg Gln Ala Arg Ala Pro Pro Ala Asp Ile1 5 10 15Val
Phe Ser Val Lys Ser Pro Pro Ser Ala Gly Tyr Leu Val Met Val20
25
30Ser Arg Gly Ala Leu Ala Asp Glu Pro Pro35 40
* * * * *
References